Hydrolytically stable maleimide-terminated polymers

ABSTRACT

The present invention is directed to hydrolytically stabilized maleimide-functionalized water soluble polymers and to methods for making and utilizing such polymers and their precursors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. provisionalapplication Ser. No. 60/437,211, filed Dec. 31, 2002, which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to particular maleimide-terminated water solublepolymers, and to methods for making and utilizing such polymers. Inparticular, the invention relates to: (i) hydrolytically stabilizedpolymers having one or more terminal maleimide groups, (ii) conjugatesformed from the attachment of a maleimide-terminated water solublepolymer reagent as described herein to another substance, such as anactive agent or surface, (iii) methods for synthesizing such polymericreagents, (iv) compositions comprising the polymeric reagents, and thelike.

BACKGROUND OF THE INVENTION

Due to recent advances in biotechnology, therapeutic proteins and otherbiomolecules, e.g. antibodies and antibody fragments, can now beprepared on a large scale, making such biomolecules more widelyavailable. Unfortunately, the clinical usefulness of potentialtherapeutic biomolecules is often hampered by their rapid proteolyticdegradation, low bioavailability, instability upon manufacture, storageor administration, or by their immunogenicity. Due to the continuedinterest in administering proteins and other biomolecules fortherapeutic use, various approaches to overcoming these deficiencieshave been explored.

One such approach which has been widely explored is the modification ofproteins and other potentially therapeutic molecules by covalentattachment of a water soluble polymer such as polyethylene glycol or“PEG” (Abuchowski, A., et al, J. Biol. Chem. 252 (11), 3579 (1977);Davis, S., et al., Clin. Exp Immunol., 46, 649-652 (1981). Thebiological properties of PEG-modified proteins, also referred to asPEG-conjugates or pegylated proteins, have been shown, in many cases, tobe considerably improved over those of their non-pegylated counterparts(Herman, et al., Macromol. Chem. Phys., 195, 203-209 (1994).Polyethylene glycol-modified proteins have been shown to possess longercirculatory times in the body due to increased resistance to proteolyticdegradation, and also to possess increased thermostability (Abuchowski,A., et al., J. Biol. Chem., 252, 3582-3586 (1977). A similar increase inbioefficacy is observed with other biomolecules, e.g. antibodies andantibody fragments (Chapman, A., Adv. Drug Del. Rev. 54, 531-545(2002)).

Typically, attachment of polyethylene glycol to a drug or other surfaceis accomplished using an activated PEG derivative, that is to say, a PEGhaving at least one activated terminus suitable for reaction with anucleophilic center of a biomolecule (e.g., lysine, cysteine and similarresidues of proteins). Most commonly employed are methods based upon thereaction of an activated PEG with protein amino groups, such as thosepresent in the lysine side chains of proteins. Polyethylene glycolhaving activated end groups suitable for reaction with the amino groupsof proteins include PEG-aldehydes (Harris, J. M., Herati, R. S., PolymPrepr. (Am. Chem. Soc., Div. Polym. Chem), 32(1), 154-155 (1991), mixedanhydrides, N-hydroxysuccinimide esters, carbonylimadazolides, andchlorocyanurates (Herman, S., et al., Macromol. Chem. Phys. 195, 203-209(1994)). Although many proteins have been shown to retain activityduring PEG modification, in some instances, polymer attachment throughprotein amino groups can be undesirable, such as when derivatization ofspecific lysine residues inactivates the protein (Suzuki, T., et al.,Biochimica et Biophysica Acta 788, 248-255 (1984)). Moreover, since mostproteins possess several available/accessible amino groups, the polymerconjugates formed are typically mixtures of mono-pegylated,di-pegylated, tri-pegylated species and so on, which can be difficultand also time-consuming to characterize and separate. Further, suchmixtures are often not reproducibly prepared, which can create problemsduring scale-up for regulatory approval and subsequentcommercialization.

One method for avoiding these problems is to employ a site-selectivepolymer reagent that targets functional groups other than amines. Oneparticularly attractive target is the thiol group on proteins, presentin the amino acid, cysteine. Cysteines are typically less abundant inproteins than lysines, thus reducing the likelihood of proteindeactivation upon conjugation to these thiol-containing amino acids.Moreoever, conjugation to cysteine sites can often be carried out in awell-defined manner, leading to the formation of single speciespolymer-conjugates.

Polyethylene glycol derivatives having a thiol-selective reactive endgroup include maleimides, vinyl sulfones, iodoacetamides, thiols, anddisulfides, with maleimides being the most popular. These derivativeshave all been used for coupling to the cysteine side chains of proteins(Zalipsky, S. Bioconjug. Chem. 6, 150-165 (1995); Greenwald, R. B. etal. Crit. Rev. Ther. Drug Carrier Syst. 17, 101-161 (2000); Herman, S.,et al., Macromol. Chem. Phys. 195, 203-209 (1994)). However, many ofthese reagents have not been widely exploited due to the difficulty intheir synthesis and purification.

As discussed above, polyethylene glycol derivatives having a terminalmaleimide group are one of the most popular types ofsulfhydryl-selective reagents, and are commercially available from anumber of sources. Although not widely appreciated or recognized, theApplicants have recognized that many PEG-maleimides unfortunately arehydrolytically unstable during storage and conjugation to a drugcandidate. More particularly, a substantial degree of hydrolysis of themaleimide ring has been observed, both prior to and after conjugation.This instability can result in the formation of multiple species of drugconjugates within a drug-conjugate composition. The various drugconjugate species are likely to possess similar biological activities,but may differ in their pharmacokinetic properties, making suchcompositions undesirable for patient administration. Additionally,separation of the open-ring and closed-ring forms of the drug conjugatecan be extremely difficult to carry out. Moreover, such hydrolyticinstability can lead to inconsistency in drug batches. Thus, theapplicants have realized a continuing need in the art for thedevelopment of new activated PEGs useful for coupling to biologicallyactive molecules, desirably in a site-selective fashion, that are stableduring both storage and coupling. This invention meets those needs.

SUMMARY OF THE INVENTION

The present invention provides a unique family of hydrolyticallystabilized maleimide-terminated polymers, where the polymers compriseparticular linkers interposed between a polymer segment and a maleimidegroup.

The invention is based on the discovery that the incorporation of asaturated acyclic, cyclic, or alicyclic hydrocarbon linker adjacent tothe maleimide ring of a maleimide-terminated polymer substantiallyreduces its instability. Provided herein are polymers having ahydrolytically stabilized maleimide ring, their polymer precursors,conjugates of the hydrolytically stabilized maleimide-terminatedpolymers, and methods for making and using such polymers and theirconjugates.

Generally, the present invention is directed to a water soluble polymerhaving the structure:

In the generalized structure above, POLY is a water soluble polymersegment, and L is a linkage that imparts hydrolytical stability to theadjacent maleimide ring. Typically the linker comprises a saturatedacyclic, cyclic or alicyclic hydrocarbon chain adjacent to the maleimidering and contains a total of about 3 to about 20 carbon atoms,optionally containing other non-interfering atoms or functional groups.

More particularly, in one aspect, the invention is directed to awater-soluble polymer having the structure:

In structure II, POLY is a water-soluble polymer segment, b is 0 or 1,and X is a hydrolytically stable linker comprising at least 3 contiguoussaturated carbon atoms. Preferably, the polymer is absent aromaticgroups and ester linkages. In one embodiment, POLY is directlycovalently bonded to the amide carbonyl carbon, optionally via anintervening oxygen ([O]_(b)) to form a carbamate group. In analternative embodiment, POLY is connected to the amide carbonyl carbon,optionally via an intervening oxygen, ([O]) in the instance when b=1,via an intervening spacer, for example, a methylene.

In one embodiment, X is a saturated acyclic, cyclic or alicyclichydrocarbon chain having a total of about 3 to about 20 carbon atoms.More particularly, X can possess a total number of carbon atoms selectedfrom the group consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, and 20. Preferred ranges for total number of carbonatoms in the linker X are from about 3 to about 20, or from about 4 toabout 12, or from about 4 to about 10, or from about 5 to about 8 atoms.

The linker X in formula II may possess any of a number of structuralfeatures. In one embodiment, X is a linear saturated acyclic hydrocarbonchain. In yet another embodiment, X is a branched saturated acyclichydrocarbon chain and can contain one or even two substituents, at anyone or more of the carbon positions in the chain. For example, X can bebranched at the carbon α to the maleimidyl group, or at the carbon β tothe maleimidyl group, or at the carbon γ to the maleimidyl group. Forhydrocarbon chains having up to 19 carbon atoms, any one of positions 1to 19 (with position 1 being the one proximal to the maleimide ring) maybe branched. For instance, for an exemplary saturated hydrocarbon chainhaving from 2 to 19 carbon atoms designatedC₁-C₂-C₃-C₄-C₅-C₆-C₇-C₈-C₉-C₁₀-C₁₁-C₁₂-C₁₃-C₁₄-C₁₅-C₁₆-C₁₇-C₁₈-C₁₉-, anyone or more of carbons C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, or C₁₉, depending on the total numberof carbons in the chain, may be branched. Preferably, in any givensaturated hydrocarbon chain or alicyclic linker, 4 or fewer carbon atomsare branched, with the overall number of branching positions preferablyequal to 1, 2, 3, or 4. Embodiments wherein the “branch” points takentogether form a saturated ring or ring system (e.g., bicyclic,tricyclic, etc.) are discussed separately below.

Representative polymers in accordance with different embodiments of theinvention are provided below.

For example, in structure III, y is an integer from 1 to about 20; andR¹ and R² in each occurrence are each independently H or an organicradical that is selected from the group consisting of alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, alkylenecycloalkyl, andsubstituted alkylenecycloalkyl.

Preferably, in structure III above, R¹ and R² in each occurrence areeach independently H or an organic radical selected from the groupconsisting of lower alkyl and lower cycloalkyl. Y is preferably selectedfrom the group consisting of 3, 4, 5, 6, 7, 8, 9, and 10. In aparticular embodiment of structure III, R¹ and R² are both H.

Various embodiments of structure III include the following.

In illustrative structure III-A, at least one of R¹ or R² on C_(α) isselected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl, and y is at least one and may possess any of theabove-described specific values.

Particular embodiments of structure III-A include those where:

(i) each of R¹ and R² on C_(α) is independently selected from the groupconsisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl,and/or

(ii) all other non-C_(α) R¹ and R² variables are H, and/or

(iii) at least one of R¹ or R² on C_(α) is lower alkyl or lowercycloalkyl, and/or

(iv) R² on C_(α) is H, and/or

(v) R¹ on C_(α) is selected from the group consisting of methyl, ethyl,propyl, isopropyl, butyl, isobutyl, pentyl, cyclopentyl, hexyl, andmethylenecyclohexyl.

Yet another particular embodiment of this aspect of the invention isprovided as structure III-B below.

wherein R¹ and R² are each independently alkyl or cycloalkyl.Alternatively, R¹ is alkyl or cycloalkyl and R² is H. Additionalembodiments of structure III-B are those where (i) R¹ and R² are eachindependently either methyl or ethyl, and/or R¹ and R² are the same.

In yet another embodiment of structure III, a polymer of the inventionpossesses the following structure:

where R¹ and R² are each independently selected from the groupconsisting of H, alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl, butare not both H, and y is at least 2.

Particular embodiments of this structure include those where (i) R¹ andR² are each independently H, lower alkyl or lower cycloalkyl, and/or(ii) R¹ and R² are each independently selected from the group consistingof H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl,cyclopentyl, hexyl, and cyclohexyl, and/or R² is H.

In yet another embodiment of structure III,

at least one of R¹ and R² attached to C_(γ) is selected from the groupconsisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl.Specific embodiments include those where: (i) at least one of R¹ and R²attached to C_(γ) is alkyl or cycloalkyl and all other R¹ and R²variables are H, and/or (ii) one of the R¹ variables attached to C_(α)or C_(β) is alkyl or cycloalkyl, and all other R¹ and R² variables areH.

As described previously, X can be a saturated cyclic or alicyclichydrocarbon chain, that is to say, the linker X may contain one or morecyclic hydrocarbons. Generally, also provided herein are polymers havingthe structure:

In the preceding structure, CYC_(a) is a cycloalkylene group having “a”ring carbons, where the value of “a” ranges from 3 to 12; and p and qare each independently 0 to 20, and p+q+a≦20. R¹ and R², in eachoccurrence, are each independently H or an organic radical that isselected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl.

CYC_(a) in accordance with the invention encompasses unicyclic,bicyclic, tricyclic structures and the like.

Various embodiments of structure IV above include those where:

(i) p and q are each independently selected from the group consisting of0, 1, 2, 3, 4, 5, 6, 7, and 8, and/or

(ii) R¹, in each occurrence, is independently H or an organic radicalthat is either lower alkyl or lower cycloalkyl, and R², in eachoccurrence, is independently H or an organic radical that is eitherlower alkyl or lower cycloalkyl, and/or

(iii) a is selected from the group consisting of 5, 6, 7, 8 and 9,and/or

(iv) a is 6 and CYC_(a) is a 1,1-, 1,2-, 1,3- or 1,4-substitutedcyclohexyl ring, and/or

(v) p and q each independently range from 0 to 4, and/or

(vi) R¹ and R² are H in every occurrence.

For linkers comprising a cyclcoalkylene group and two substituentsthereon, the substituents can be either cis or trans.

Specific embodiments of structure IV include:

wherein q and p are as described above. In a particular embodiment, qand p each independently range from 0 to 6. In yet another embodiment, qranges from 0 to 6 and p is zero.

Yet another exemplary polymer structure having a cycloalkylene ring inaccordance with the invention is:

wherein q and p are as defined above, and more preferably, eachindependently range from 0 to 6.

Polymers of the invention include monofunctional, bifunctional, andmulti-functional structures.

For instance, a polymer of the invention may be described generally bythe following structure:

where X and b are as previously defined, b′ is 0 or 1, and X′ is ahydrolytically stable linker comprising at least 3 contiguous saturatedcarbon atoms. In the above embodiment, b and b′ may be the same ofdifferent, and X and X′ may be the same or different. In one particularembodiment the polymer reagent is homo-bifunctional, that is to say,both reactive end groups are the same. In this instance, b equals b′ andX equals X′.

Preferably, the water-soluble polymer segment in any of the polymermaleimides provided herein is a poly(alkylene oxide), a poly(vinylpyrrolidone), a poly(vinyl alcohol), a polyoxazoline, apoly(acryloylmorpholine), or a poly(oxyethylated polyol). In a preferredembodiment, the polymer segment is a poly(alkylene oxide), preferablypoly(ethylene glycol).

According to one embodiment, the poly(ethylene glycol) segment comprisesthe structure: Z-(CH₂CH₂O)_(n)—CH₂CH₂—, where n ranges from about 10 toabout 4000 and Z is a moiety comprising a functional group selected fromthe group consisting of hydroxy, amino, ester, carbonate, aldehyde,alkenyl, acrylate, methacrylate, acrylamide, sulfone, thiol, carboxylicacid, isocyanate, isothiocyanate, hydrazide, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, alkoxy, benzyloxy, silane,lipid, phospholipid, biotin, and fluorescein. In this embodiment, Zcomprises a reactive functional group or an end-capping group.

In yet a more specific embodiment, POLY may be terminally capped with anend-capping moiety such as alkoxy, substituted alkoxy, alkenyloxy,substituted alkenyloxy, alkynyloxy, substituted alkynyloxy, aryloxy,substituted aryloxy, or phospholipid. Preferred end-capping groupsinclude methoxy, ethoxy, and benzyloxy.

Generally, POLY possesses a nominal average molecular mass fallingwithin one of the following ranges: from about 100 daltons to about100,000 daltons, from about 1,000 daltons to about 50,000 daltons, orfrom about 2,000 daltons to about 30,000 daltons. Preferred molecularmasses for POLY include 250 daltons, 500 daltons, 750 daltons, 1 kDa, 2kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 30 kDa, 40 kDa, and 50 kDa, or evengreater.

The polymer segment may possess any of a number of geometries, e.g., maybe linear, branched or forked.

The polymer of the invention may be multi-armed. An exemplary multi-armpolymer in accordance with the invention has the structure:

In the above illustrative structure, d is an integer from 3 to about100, and R is a residue of a central core molecule having 3 or morehydroxyl groups, amino groups, or combinations thereof. Preferably, d isan integer from 3 to about 12.

In an alternative multi-arm embodiment, the polymer corresponds to thestructure:

In this structure,

PEG is —(CH₂CH₂O)_(n)CH₂CH₂—,

M is:

and m is selected from the group consisting of 3, 4, 5, 6, 7, and 8.

In yet another aspect, provided herein are polymers having the abovedescribed features and exemplary structures, with the exception that themaleimide group in the above-described polymers is replaced with anamino group, preferably a primary amino, —NH₂. Such polymers are usefulnot only as activated polymer reagents, e.g., for conjugation to anactive agent, but are also precursors to the stabilized maleimidepolymers of the invention.

For example, the invention encompasses a water-soluble polymer inaccordance with the structure:

where the variables X and b are as previously described, both generallyand in specific embodiments. Preferably, the polymer is absent aromaticgroups and ester linkages.

In yet another aspect, the invention provides a water soluble polymerhaving the structure:

where POLY is a water-soluble polymer segment, and X is a hydrolyticallystable linker that is a saturated cyclic or alicyclic hydrocarbon chainhaving a total of about 3 to about 20 carbon atoms. Preferably, thepolymer is absent aromatic groups and ester linkages.

Polymers in accordance with this aspect of the invention encompass, invarious embodiments, those where X corresponds to the general andspecific cyclic and alicyclic hydrocarbon structures described herein.

In one particular embodiment of structure XIII, the linker X has thestructure:

-   -   where        -   CYC_(a) is a cycloalkylene group having “a” ring carbons,            where the value of “a” ranges from 3 to 12; p and q are each            independently 0 to 20, and p+q+a≦20. In structure XIII-A,            each of R¹ and R², in each occurrence, is independently H or            an organic radical that is selected from the group            consisting of alkyl, substituted alkyl, cycloalkyl,            substituted cycloalkyl, alkylenecycloalkyl, and substituted            alkylenecycloalkyl. In instances where CYC_(a) possesses            only two substituents, such substituents can be cis or            trans.

In yet another embodiment of structure XIII-A, p and q are eachindependently selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,7, and 8.

In yet another embodiment of structure XIII-A, R¹, in each occurrence,is independently H or an organic radical that is selected from the groupconsisting of lower alkyl, lower cycloalkyl, and loweralkylenecycloalkyl, and R², in each occurrence, is independently H or anorganic radical that is selected from the group consisting of loweralkyl, lower cycloalkyl, and lower alkylenecycloalkyl.

In yet another embodiment of structure XIII-A, a is selected from thegroup consisting of 5, 6, 7, 8 and 9.

In a preferred embodiment of structure XIII-A, a is 6 and CYC_(a) is a1,1-, 1,2-, 1,3- or 1,4-substituted cyclohexyl ring. Additionalembodiments include those where p and q each independently range from 0to 4, and/or where R¹ and R² are H in every occurrence.

Particularly, certain embodiments of structure XIII include:

-   1

wherein q and p each independently range from 0 to 6.

In yet another embodiment of structure XIII, CYC_(a) is bicyclic ortricyclic.

In yet another aspect, the invention includes hydrogels prepared usingany one or more of the polymers described herein.

In yet another aspect, provided is a method for forming a hydrolyticallystable maleimide-terminated polymer. The method includes the steps of(a) reacting a polymer having the structure, POLY-[O]_(b)—C(O)-LG (IX),with a diamine having the structure, NH₂—X—NH₂ (XII), under conditionseffective to form POLY-[O]_(b)—C(O)—HN—X—NH₂ (X), followed by (b)converting POLY-[O]_(b)—C(O)—HN—X—NH₂ (X) intoPOLY-[O]_(b)—C(O)—HN—X-MAL (II).

The variables POLY, b and X are as described previously, both generallyand specifically, LG represents a leaving group, and MAL is maleimide.Preferably, the resulting product, POLY-[O]_(b)—C(O)—HN—X-MAL, is absentaromatic groups and ester linkages.

The method can be used to prepare any of the polymer-terminatedmaleimides described herein.

Preferred leaving groups include halide, N-hydroxysuccinimide,N-hydroxybenzotriazole, para-nitrophenolate.

In one embodiment of the method, one of the amino groups in saidNH₂—X—NH₂ reagent is in protected form. In this instance, the methodwill generally comprise, after the reacting step, deprotecting the aminogroup in POLY-[O]_(b)—C(O)—H₂N—X—NH₂.

The reacting step is typically carried out in an organic solvent.Typical solvents include acetonitrile, chlorinated hydrocarbons,aromatic hydrocarbons, tetrahydrofuran (THF), dimethylformamide (DMF),and dimethylsulfoxide.

In another embodiment of the method, the reacting step is conductedunder an inert atmosphere such as nitrogen or argon.

Temperatures for carrying out the reacting step range from about 0 to100° C.

In a further embodiment, the reacting step is carried out in thepresence of a base. Examplary bases include triethyl amine and othersimilar tertiary amines, pyridine, 4-(dimethylamino)pyridine, andinorganic bases such as sodium carbonate.

In a preferred embodiment, the method further includes the step ofpurifying the product from step (a) prior to the converting step, forexample, by column chromatography, preferably by ion exchangechromatography.

In yet another specific embodiment, the converting step comprisesreacting POLY-[O]_(b)—C(O)—H₂N—X—NH₂ with a reagent selected from thegroup consisting of N-methoxycarbonylmaleimide,exo-7-oxa[2.2.1]bicycloheptane-2,3-dicarboxylic anhydride, and maleicanhydride, under conditions suitable for formingPOLY-[O]_(b)—C(O)—H₂N—X-MAL in a reaction mixture.

In an embodiment where the reagent is N-methoxycarbonylmaleimide, theconverting step is preferably carried out in water or an aqueous mixtureof water and a water miscible solvent such as acetone or acetonitrile.

In an embodiment of the above method where the reagent is maleicanhydride, the converting step comprises reactingPOLY-[O]_(b)—C(O)—H₂N—X—NH₂ with maleic anhydride under conditionseffective to form POLY-[O]_(b)—C(O)—NH—X—NH—C(O)CH═CHCOOH (XI) as anintermediate, followed by heatingPOLY-[O]_(b)—C(O)—H₂N—X—NH—C(O)CH═CHCOOH under conditions effective topromote cyclization by elimination of water to formPOLY-[O]_(b)—C(O)—NH—X-MAL.

Generally, the method further comprises the step of recovering theproduct, POLY-[O]_(b)—C(O)—H₂N—X-MAL, from the reaction mixture.

Preferably, the recovered product has a purity of greater than about80%, and is absent polymeric impurities other than the desired product.

Exemplary diamines for carrying out the method include

where the variables encompass those both generally and specificallydescribed above.

In yet another aspect, provided herein is an alternative method forpreparing a hydrolytically stable maleimide-terminated polymer of theinvention. The method includes the steps of reactingPOLY-[O]_(b)—C(O)-LG (IX) with H₂N—X-MAL (XIV) under conditionseffective to form POLY-[O]_(b)—C(O)—HN—X-MAL (II), where the variablesPOLY, b, X, LG, and MAL are as previously defined, both generally andspecifically, regardless of the subject embodiment used forexemplification purposes.

In yet another aspect, the invention provides a conjugate formed byreaction of a biologically active agent with any of the herein describedhydrolytically stable maleimide- or amino-terminated polymers.

More particularly, one embodiment of this aspect of the inventionincludes a conjugate comprising the following structure:

where the variables POLY, b and X are as previously defined, bothgenerally and specifically, regardless of the subject exemplifyingembodiment, “POLY-[O]_(b)—C(O)—NH—X—” is absent aromatic groups andester linkages, and “—S-biologically active agent” represents abiologically active agent comprising a thiol (—SH) group.

In one embodiment, provided is a composition comprising the aboveconjugate. In a more particular embodiment, the conjugate compositioncomprises a single polymer conjugate species.

In yet another embodiment, the invention is directed to a conjugatecomprising the following structure:

wherein POLY, b, and X are as defined above, both generally andspecifically, “POLY-[O]_(b)—C(O)—NH—X—” is absent aromatic groups andester linkages, and “—NH— biologically active agent” represents abiologically active agent comprising an amino group.

In yet another related aspect, the invention provides a method forforming a polymer conjugate, where the method includes the step ofcontacting a biologically active agent comprising a reactive thiolgroup, “HS-biologically active agent”, with a hydrolytically ring stablemaleimide terminated polymer of the invention, under conditionseffective to promote formation of a polymer conjugate having thestructure:

These and other objects and features of the invention will become morefully apparent when read in conjunction with the following figures anddetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B provide structures of exemplary polymer maleimides ofthe invention containing hydrolytically stable cyclic (includingbicyclic and tricyclic) linkers, and

FIG. 2 provides structures of exemplary diamines useful in preparingcertain stabilized polymer maleimides of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to the particularpolymers, synthetic techniques, active agents, and the like as such mayvary. It is also to be understood that the terminology used herein isfor describing particular embodiments only, and is not intended to belimiting.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

Definitions

The following terms as used herein have the meanings indicated.

As used in the specification, and in the appended claims, the singularforms “a”, “an”, “the”, include plural referents unless the contextclearly dictates otherwise.

“PEG” or “poly(ethylene glycol)” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Typically, PEGs for use in thepresent invention will comprise one of the two following structures:“—(CH₂CH₂O)_(n)—” or “—(CH₂CH₂O)_(n-1)CH₂CH₂—,” depending upon whetheror not the terminal oxygen(s) has been displaced, e.g., during asynthetic transformation. The variable (n) ranges from 3 to 3000, andthe terminal groups and architecture of the overall PEG may vary. WhenPEG further comprises a linker moiety (to be described in greater detailbelow), the atoms comprising the linker, when covalently attached to aPEG segment, do not result in formation of (i) an oxygen-oxygen bond(—O—O—, a peroxide linkage), or (ii) a nitrogen-oxygen bond (N—O, O—N).“PEG” means a polymer that contains a majority, that is to say, greaterthan 50%, of subunits that are —CH₂CH₂O—. PEGs for use in the inventioninclude PEGs having a variety of molecular weights, structures orgeometries (e.g., branched, linear, forked PEGs, dendritic, and thelike), to be described in greater detail below.

“PEG diol”, also known as alpha-, omega-dihydroxylpoly(ethylene glycol),can be represented in brief form as HO-PEG-OH, where PEG is as definedabove.

“Water-soluble”, in the context of a polymer of the invention or a“water-soluble polymer segment” is any segment or polymer that issoluble in water at room temperature. Typically, a water-soluble polymeror segment will transmit at least about 75%, more preferably at leastabout 95% of light, transmitted by the same solution after filtering. Ona weight basis, a water-soluble polymer or segment thereof willpreferably be at least about 35% (by weight) soluble in water, morepreferably at least about 50% (by weight) soluble in water, still morepreferably about 70% (by weight) soluble in water, and still morepreferably about 85% (by weight) soluble in water. It is most preferred,however, that the water-soluble polymer or segment is about 95% (byweight) soluble in water or completely soluble in water.

An “end-capping” or “end-capped” group is an inert or non-reactive grouppresent on a terminus of a polymer such as PEG. An end-capping group isone that does not readily undergo chemical transformation under typicalsynthetic reaction conditions. An end capping group is generally analkoxy group, —OR, where R is an organic radical comprised of 1-20carbons and is preferably lower alkyl (e.g., methyl, ethyl) or benzyl.“R” may be saturated or unsaturated, and includes aryl, heteroaryl,cyclo, heterocyclo, and substituted forms of any of the foregoing. Forinstance, an end capped PEG will typically comprise the structure“RO—(CH₂CH₂O)_(n)—”, where R is as defined above. Alternatively, theend-capping group can also advantageously comprise a detectable label.When the polymer has an end-capping group comprising a detectable label,the amount or location of the polymer and/or the moiety (e.g., activeagent) to which the polymer is coupled, can be determined by using asuitable detector. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g.,dyes), metal ions, radioactive moieties, and the like. The end-cappinggroup can also advantageously comprise a phospholipid. When the polymerhas an end-capping group such as a phospholipid, unique properties (suchas the ability to form organized structures with similarly end-cappedpolymers) are imparted to the polymer. Exemplary phospholipids include,without limitation, those selected from the class of phospholipidscalled phosphatidylcholines. Specific phospholipids include, withoutlimitation, those selected from the group consisting ofdilauroylphosphatidylcholine, dioleylphosphatidylcholine,dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,behenoylphosphatidylcholine, arachidoylphosphatidylcholine, andlecithin.

“Non-naturally occurring” with respect to a polymer of the inventionmeans a polymer that in its entirety is not found in nature. Anon-naturally occurring polymer of the invention may however contain oneor more subunits or segments of subunits that are naturally occurring,so long as the overall polymer structure is not found in nature.

“Molecular mass” in the context of a water-soluble polymer of theinvention such as PEG, refers to the nominal average molecular mass of apolymer, typically determined by size exclusion chromatography, lightscattering techniques, or intrinsic velocity determination in1,2,4-trichlorobenzene. The polymers of the invention are typicallypolydisperse, possessing low polydispersity values of less than about1.20.

The term “reactive” or “activated” refers to a functional group thatreacts readily or at a practical rate under conventional conditions oforganic synthesis. This is in contrast to those groups that either donot react or require strong catalysts or impractical reaction conditionsin order to react (i.e., a “nonreactive” or “inert” group).

“Not readily reactive” or “inert” with reference to a functional grouppresent on a molecule in a reaction mixture, indicates that the groupremains largely intact under conditions effective to produce a desiredreaction in the reaction mixture.

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group will vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups whichmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as a p-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

A functional group in “protected form” refers to a functional groupbearing a protecting group. As used herein, the term “functional group”or any synonym thereof is meant to encompass protected forms thereof.

The term “linker” is used herein to refer to an atom or a collection ofatoms optionally used to link interconnecting moieties, such as apolymer segment and a maleimide. The linkers of the invention aregenerally hydrolytically stable.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa relatively weak bond that reacts with water (i.e., is hydrolyzed)under physiological conditions. The tendency of a bond to hydrolyze inwater will depend not only on the general type of linkage connecting twocentral atoms but also on the substituents attached to these centralatoms. Appropriate hydrolytically unstable or weak linkages include butare not limited to carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides andoligonucleotides, thioesters, thiolesters, and carbonates.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or linker, for the purposes of thepresent invention, and in particular in reference to the polymers of theinvention, refers to an atom or to a collection of atoms, that ishydrolytically stable under normal physiological conditions. That is tosay, a hydrolytically stable linkage does not undergo hydrolysis underphysiological conditions to any appreciable extent over an extendedperiod of time. Examples of hydrolytically stable linkages include butare not limited to the following: carbon-carbon bonds (e.g., inaliphatic chains), ethers, amides, urethanes, amines, and the like.Hydrolysis rates of representative chemical bonds can be found in moststandard chemistry textbooks.

A “hydrolytically stabilized maleimide ring”, in reference to a polymerof the invention, is one that resists hydrolysis of the maleimide ringin comparison to the ring opening-stability of its linkerless polymermaleimide counterpart. For example, if a subject water soluble maleimidehas a structureCH₃O—(CH₂CH₂O)_(5K)—CH₂CH₂—C(O)—NH—CH₂-1,3-C₆H₁₀—CH₂-MAL, where thelinker is —C(O)—NH—CH₂-1,3-C₆H₁₀—CH₂—, then the corresponding linkerlessversion to form a basis for comparison isCH₃O—(CH₂CH₂O)_(5K)—CH₂CH₂-MAL. Typically, such hydrolysis evaluationsare carried out at pH 7.5 in phosphate buffer at room temperature andare measured by observing the UV absorption of the maleimide ring. So, ahydrolytically stabilized maleimide ring contained in a polymer reagentof the invention is one that has a degree of hydrolytic stability thatis improved over that of its linkerless counterpart. Preferably, ahydrolytically stabilized maleimide ring in accordance with theinvention results in a stabilized polymer maleimide having a hydrolysishalf-life under the above-described conditions of at least about 16hours, and more preferably of at least about 20 hours.

“Branched” in reference to the geometry or overall structure of apolymer refers to polymer having 2 or more polymer “arms”. A branchedpolymer may possess 2 polymer arms, 3 polymer arms, 4 polymer arms, 6polymer arms, 8 polymer arms or more. One particular type of highlybranched polymer is a dendritic polymer or dendrimer, that for thepurposes of the invention, is considered to possess a structure distinctfrom that of a branched polymer.

“Branch point” refers to a bifurcation point comprising one or moreatoms at which a polymer splits or branches from a linear structure intoone or more additional polymer arms.

A “dendrimer” is a globular, size monodisperse polymer in which allbonds emerge radially from a central focal point or core with a regularbranching pattern and with repeat units that each contribute a branchpoint. Dendrimers exhibit certain dendritic state properties such ascore encapsulation, making them unique from other types of polymers.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95% or greater of some given quantity.

An “alkyl” or “alkylene” group, depending upon its position in amolecule and the number of points of attachment of the group to atomsother than hydrogen, refers to a hydrocarbon chain or moiety, typicallyranging from about 1 to 20 atoms in length. Such hydrocarbon chains arepreferably but not necessarily saturated unless so indicated and may bebranched or straight chain, although typically straight chain ispreferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl,pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, hexyl, heptyl, andthe like.

“Lower alkyl” or “lower alkylene” refers to an alkyl or alkylene groupas defined above containing from 1 to 6 carbon atoms, and may bestraight chain or branched, as exemplified by methyl, ethyl, n-butyl,i-butyl, t-butyl.

“Cycloalkyl” or “cycloalkylene”, depending upon its position in amolecule and the number of points of attachment to atoms other thanhydrogen, refers to a saturated or unsaturated cyclic hydrocarbon chain,including polycyclics such as bridged, fused, or spiro cyclic compounds,preferably made up of 3 to about 12 carbon atoms, more preferably 3 toabout 8.

“Lower cycloalkyl” or “lower cycloalkylene” refers to a cycloalkyl groupcontaining from 1 to 6 carbon atoms.

“Alicyclic” refers to any aliphatic compound that contains a ring ofcarbon atoms. An alicyclic group is one that contains a “cycloalkyl” or“cycloalkylene” group as defined above that is substituted with one ormore alkyl or alkylenes.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenon-interfering substituents, such as, but not limited to: C₃—C₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl;substituted phenyl; and the like. For substitutions on a phenyl ring,the substituents may be in any orientation (i.e., ortho, meta, or para).

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁—C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy,benzyl, etc.), preferably C₁—C₇.

As used herein, “alkenyl” refers to a branched or unbranched hydrocarbongroup of 1 to 15 atoms in length, containing at least one double bond,such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, and the like.

The term “alkynyl” as used herein refers to a branched or unbranchedhydrocarbon group of 2 to 15 atoms in length, containing at least onetriple bond, ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl,octynyl, decynyl, and so forth.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably N, O, or S, or a combination thereof. Heteroaryl rings mayalso be fused with one or more cyclic hydrocarbon, heterocyclic, aryl,or heteroaryl rings.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom which is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heteroaryl” is heteroaryl having one or morenon-interfering groups as substituents.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from non-interfering substituents.

“Electrophile” refers to an ion, atom, or collection of atoms that maybe ionic, having an electrophilic center, i.e., a center that iselectron seeking, capable of reacting with a nucleophile.

“Nucleophile” refers to an ion or atom or collection of atoms that maybe ionic, having a nucleophilic center, i.e., a center that is seekingan electrophilic center, and capable of reacting with an electrophile.

“Active agent” as used herein means any agent, drug, compound,composition of matter or mixture which provides some pharmacologic,often beneficial, effect that can be demonstrated in-vivo or in vitro.This includes foods, food supplements, nutrients, nutriceuticals, drugs,vaccines, antibodies, vitamins, and other beneficial agents. As usedherein, these terms further include any physiologically orpharmacologically active substance that produces a localized or systemiceffect in a patient.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to an excipient that can be included in the compositionsof the invention and that causes no significant adverse toxicologicaleffects to the patient.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a PEG-active agent conjugate present in apharmaceutical preparation that is needed to provide a desired level ofactive agent and/or conjugate in the bloodstream or in the targettissue. The precise amount will depend upon numerous factors, e.g., theparticular active agent, the components and physical characteristics ofpharmaceutical preparation, intended patient population, patientconsiderations, and the like, and can readily be determined by oneskilled in the art, based upon the information provided herein andavailable in the relevant literature.

“Multi-functional” in the context of a polymer of the invention means apolymer backbone having 3 or more functional groups contained therein,where the functional groups may be the same or different, and aretypically present on the polymer termini. Multi-functional polymers ofthe invention will typically contain from about 3-100 functional groups,or from 3-50 functional groups, or from 3-25 functional groups, or from3-15 functional groups, or from 3 to 10 functional groups, or willcontain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups within the polymerbackbone.

A “difunctional” polymer means a polymer having two functional groupscontained therein, typically at the polymer termini. When the functionalgroups are the same, the polymer is said to be homodifunctional. Whenthe functional groups are different, the polymer is said to beheterobifunctional

A basic or acidic reactant described herein includes neutral, charged,and any corresponding salt forms thereof.

“Polyolefinic alcohol” refers to a polymer comprising an olefin polymerbackbone, such as polyethylene, having multiple pendant hydroxyl groupsattached to the polymer backbone. An exemplary polyolefinic alcohol ispolyvinyl alcohol.

As used herein, “non-peptidic” refers to a polymer backbonesubstantially free of peptide linkages. However, the polymer may includea minor number of peptide linkages spaced along the repeat monomersubunits, such as, for example, no more than about 1 peptide linkage perabout 50 monomer units.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of apolymer of the invention, typically but not necessarily in the form of apolymer-active agent conjugate, and includes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

By “residue” is meant the portion of a molecule remaining after reactionwith one or more molecules. For example, a biologically active moleculeresidue in the polymer conjugate of the invention is the portion of abiologically active molecule remaining following covalent linkage to apolymer backbone.

The term “conjugate” is intended to refer to the entity formed as aresult of covalent attachment of a molecule, e.g., a biologically activemolecule or any reactive surface, to a reactive polymer molecule,preferably a reactive poly(ethylene glycol).

The term “electron withdrawing group” refers to a chemical moiety thatbrings electron density towards itself and away from other areas of amolecule through either mesomeric mechanisms (i.e., adding or removinglocal electron density through π bonds) or inductive mechanisms (i.e.,an electronegative moiety withdrawing electron density along a σ bond,thereby polarizing the bond).

The term “steric hindrance” refers to spatial, mechanical interferencebetween two chemical groups.

Stabilized Polymer Maleimides—General Features

The present invention provides water-soluble and non-peptidic polymerswhose maleimide rings are hydrolytically stable in comparison to theirlinkerless maleimide counterparts. Generally, the feature of resistanceto hydrolysis, i.e., with regard to the maleimide ring, is imparted bythe introduction of a linker between the polymer segment and themaleimide. Typically, the linker comprises a saturated acyclic, cyclic,or alicyclic hydrocarbon chain covalently attached to, and immediatelyadjacent to, the nitrogen atom of the maleimide. The structure and sizeof the hydrocarbon chain, which may comprise alkylene groups, cycloalkylgroups, or combinations thereof, in either substituted ornon-substituted form, is designed to retard ring opening of themaleimide by i) providing sufficient distance between the maleimide andany electron withdrawing groups in the linker or the polymer segment,thereby enabling electron release to the maleimide ring, and/or ii)providing steric hindrance to the hydrolytic process. In this manner,the linkers described herein stabilize the maleimide ring againsthydrolysis and resultant ring-opening. Thus, the maleimide-terminatedpolymers of the invention exhibit greater stability, e.g., uponsynthesis, isolation, and storage, and can be conjugated to biologicallyactive molecules over a wider range of pH values without producingsignificant amounts of open-ring conjugates. Hydrolysis data providedherein is indicative of this point. The synthesis of representativestabilized polymer maleimides is described in Examples I, II, III, IV,V, IX, X, XI, and XII. As can be seen, linear acyclic, branched acyclic,and alicyclic linkers are all effective in enhancing the stability ofthe adjacent maleimide ring. Hydrolysis data illustrative of thisfeature for both the polymer reagents and their corresponding conjugatesis provided in Examples VII, VII, and XIII.

The linkers as described in greater detail below may also include one ormore non-hydrocarbon yet hydrolytically stable and non-reactive atoms orcollection of atoms, such as hydroxyl, sulfur, oxygen, and the like.

The maleimide-functionalized polymers of the invention are preferablyhydrolytically stable over a wide pH range, such as from about 5 toabout 10. In particular, most preferably, the reactive polymermaleimides of the invention are hydrolytically stable at pHs suitablefor conjugation to thiol or amino groups on biologically activemolecules, such as proteins. For example, the polymers of the inventionare preferably resistant to hydrolysis-induced maleimide ring opening(i.e., are not prone to formation of maleimic acid if unconjugated orformation of succinamidic acid if conjugated) at pHs ranging from about7 to about 10, and more preferably at pHs from about 7 to about 8.5. Asdefined herein, a hydrolytically stable maleimide is one in which thehalf-life of the maleimide at 25° C. and a pH of 7.5 in an aqueousmedium (e.g., phosphate buffer) is at least about 16 hours, morepreferably at least about 20 hours, most preferably at least about 28hours.

The half-life of a polymer maleimide can be determined by measuring theconcentration of the maleimide-terminated polymer over time using HPLCor by observing the UV absorption of the maleimide ring.

The saturated acyclic, cyclic, or alicyclic hydrocarbon linker adjacentto the maleimide group preferably has a chain length of at least 3carbon atoms and contains at least 3 contiguous carbon atoms. Morepreferably, the linker possesses at least about 4 carbon atoms, mostpreferably at least about 5 or 6 carbon atoms. The chain length ismeasured as the number of carbon atoms forming the shortest atom chainlinking the nitrogen atom of the maleimide to the polymer segment.Typically, the total number of carbon atoms in the linker includingchain substituents, ranges from 4 to about 20 atoms, preferably 4 toabout 12 atoms, more preferably 4 to about 10 atoms and most preferably5 to about 8 atoms. The invention includes linkers having, for example,4, 5, 6, 7, 8, 9, 10, 11, and 12 total carbon atoms.

General Structural Features of the Polymer Maleimide

Generally speaking, the reactive polymers of the invention possesses awater soluble polymer segment that is connected to a maleimide ring viaa hydrolytically stable linker. The hydrolytically stable linker iseffective to impart hydrolytic stability to the maleimide ring to whichit is directly covalently attached. More particularly, the polymersegment, referred to herein generally as POLY, is covalently attached tothe hydrolytically stable linker, X, via an intervening —O—, —C(O)—NH—,or O—C(O)—NH— group. X typically contains at least 3 contiguoussaturated carbon atoms. Preferably although not necessarily, theresultant polymer maleimide is absent aromatic groups and esterlinkages.

Also provided herein are polymers having both the generalized andspecific illustrative structural features described for the stabilizedpolymer maleimides, with the exception that the maleimide ring isreplaced by an amino group, preferably a primary amino group. Thus, allstructures and descriptions herein directed towards maleimide-terminatedpolymer reagents should be extended to their amino-terminatedcounterparts as described above.

The linker, X, typically contains from about 1 to about 20 carbon atoms.Generally, X is a hydrocarbon chain possessing only carbon and hydrogenatoms, however, in certain embodiments, X may contain additionalnon-reactive atoms or functional groups such as hydroxyl groups, ethers,thioethers, or other non-reactive groups. Preferably, such groups oratoms are positioned remote from the maleimide ring. More preferably,such non-reactive atoms or groups are positioned a distance of at least4 carbons from the maleimide nitrogen. Even more preferably, suchadditional non-reactive atoms or groups contained in X are positioned atleast 4 carbons, at least 5 carbons or at least 6 carbons or moredistant from the maleimide nitrogen. Such groups are more likely to becontained within cycloalkyl or alicyclic X's than in their acycliccounterparts, simply due to their presence in many commerciallyavailable starting materials.

A polymer maleimide of the invention is generally characterized by thefollowing formula:

-   -   where b is 0 or 1. In structure I, when both the carbonyl and        —NH groups are absent (i.e., each subscript is equal to zero), b        is equal to 1. Features of the linker X are described and        exemplified in greater detail in the section that follows.        The Linker Moiety

As mentioned previously, the linker, X, comprises a saturated acyclic orcyclic or alicyclic hydrocarbon moiety adjacent to the nitrogen atom ofthe maleimide ring. The size and structure of X is designed to improvethe hydrolytic stability of the maleimide ring, generally by increasingthe distance between the maleimide ring and electron withdrawing groupspresent in the molecule or by providing steric hindrance to themaleimide hydrolysis reaction. Typically, X contains a total of about 3to about 20 carbon atoms. More particularly, X can possess a totalnumber of carbon atoms selected from the group consisting of 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. Preferredranges for total number of carbon atoms in the linker X are from about 3to about 20, or from about 4 to about 12, or from about 4 to about 10,or from about 5 to about 8 atoms.

Exemplary hydrocarbon linkages include straight chain saturated acyclichydrocarbons comprising at least 3 contiguous carbon atoms, such astrimethylene, tetramethylene, pentamethylene, and hexamethylene, and soforth. That is to say, in its simplest form, X equals —(CH₂)_(y), wherey ranges from 3 to about 20. That is to say, Y may possess any of thefollowing values: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20. Alternatively, X can be branched, i.e., can contain oneor even two substituents, at any one or more of the carbon positions inthe chain. That is to say, X can be branched at the carbon α to themaleimidyl group, or at the carbon β to the maleimidyl group, or at thecarbon γ to the maleimidyl group. For hydrocarbon chains having up to 19carbon atoms, any one of positions 1 to 19 (with position 1 being theone proximal to the maleimide ring) may be branched. For instance, foran exemplary saturated hydrocarbon chain having from 2 to 19 carbonatoms designatedC₁-C₂-C₃-C₄-C₅-C₆-C₇-C₈-C₉-C₁₀-C₁₁-C₁₂-C₁₃-C₁₄-C₁₅-C₁₆-C₁₇-C₁₈-C₁₉-, anyone or more of carbons C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, or C₁₉, depending on the total numberof carbons in the chain, may be branched, that is to say, may possessone or even two substituents. Typically, a branching group is an alkylgroup, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkylenecycloalkyl, or substituted cycloalkyl. Particularly preferredcycloalkyls are cyclopentyl, cyclohexyl, cycloheptyl, and the like, andany of the previous cycloalkyls having one or more methylene groups(e.g., methylene, dimethylene, trimethylene, tetramethylene, etc.)connecting the cycloalkyl ring to the branching carbon. Preferably, inany given saturated hydrocarbon chain or alicyclic linker, 4 or fewercarbon atoms are branched, with the overall number of branchingpositions preferably equal to 1, 2, 3, or 4. Embodiments wherein the“branch” points taken together form a saturated ring or ring system(e.g., bicyclic, tricyclic, etc.) are discussed separately below. Mostpreferably, when X is branched, the branching carbon is singly branched,i.e., has one rather than two branching substituents.

For example, the linkage may possess the structure —(CR₁R₂)_(y)—,wherein R₁ and R₂ are each independently H, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl, and y is an integer from about 1 to about 20,preferably from about 3 to about 20, and even more preferably from 4 toabout 12. When X is branched, preferably the branching is at one or moreof C1, C1, C3, or C4, that is to say, the carbon atom positions closestto the maleimide ring, in order to provide maximal steric hindrance tothe maleimide ring hydrolysis reaction. (When discussing carbon atompositions within the linker, X, C1 refers to the carbon atom adjacent tothe maleimide nitrogen, and so on). When X is branched, the branchinggroups (e.g., alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl)typically contain fewer than 8 or so carbon atoms. When the branchinggroup is alkyl or cycloalkyl, preferably the alkyl group is lower alkylsuch as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, penyl,cyclopentyl, hexyl, and cyclohexyl.

As dicussed above, the linker X itself can be cyclic or alicyclic.Specifically, X may possess the form:

where CYC_(a) is a cycloalkylene group having “a” ring carbons, wherethe value of “a” ranges from 3 to 12; p and q are each independently 0to 20, and p+q+a≦20, R¹, in each occurrence, is independently H or anorganic radical that is selected from the group consisting of alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl,alkylenecycloalkyl, and substituted alkylenecycloalkyl, and R², in eachoccurrence, is independently H or an organic radical that is selectedfrom the group consisting of alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl. In a preferred embodiment, R₁ and R₂, in eachoccurrence, are both H. Preferably, p and q each independently are 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10. Even more preferably, p and q eachindependently range from 0 to 6 or 0 to 4. Preferably, the cycloalkylring represented by CYC_(a) contains from 5 to about 12 ring carbonatoms, and even more preferably from about 6 to about 10 ring carbonatoms. Representative cycloalkyl groups include C3-C8 cycloalkylene,such as cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene,and cycloheptylene, and cyclooctylene. CYC_(a) may optionally besubstituted with one or more alkyl groups, preferably lower alkylgroups, which can be at any position within the ring. In instances whereCYC_(a) is substituted with only two substituents, i.e., (CR₁R₂)_(p) and(CR₁R₂)_(q) as shown in structure IV above, the substituents cansimilarly be positioned on any one or more carbons in the ring. Forinstance, for a cyclopenylene, the substituents can be at either the1,1-, 1,2-, or 1,3-positions. For a cyclohexylene ring, the substituentscan be at the 1,1-, 1,2-, 1,3-, or 1,4-positions. Particularly preferredembodiments are those where R₁ and R₂ are H in all instances, and p andq are each independently selected from 0, 1, 2, and 3. Illustrativelinkers include 1,2-(CH₂)_(0,1,2,3)—C₆H₄—(CH₂)_(0,1,2,3), and1,3-(CH₂)_(0,1,2,3)—C₆H₄—(CH₂)_(0,1,2,3), and1,4-(CH₂)_(0,1,2,3)—C₆H₄—(CH₂)_(0,1,2,3)—. In instances in which CYC_(a)possesses only two substituents, the substituents may be either cis ortrans. When CYC_(a) contains more than two substituents, thesubstituents may be in any relative orientation to one another.

CYC_(a) also encompasses bicyclic rings. Exemplary bicyclic ringscorresponding to CYC_(a) include rings such as bicyclo[1.1.1]pentane,bicyclo[2.2.1]hexane, bicyclo[2.2.1]heptane, bicycle[2.2.2]octane,bicyclo[3.1.0]hexane, bicyclo[3.1.1]heptane, bicyclo[3.2.1]octane,bicyclo[3.3.1]nonane, bicyclo[3.3.2]decane, bicyclo[3.3.3]undecane andthe like. CYC also encompasses tricyclic ring systems such asadamantane. Some of these bi- and tricyclic systems are shown below:

These rings may possess alkylene or substituted alkylene groupscorresponding to —[C(R₁)(R₂)]_(p) and —[C(R₁)(R₂)]_(q) in any availableposition within the ring system. Moreoever, the bicyclic or tricyclicring may also possess additional substituents in addition to—[C(R₁)(R₂)]_(p) and —[C(R₁)(R₂)]_(q). Preferably, such substituents arelower alkyl, hydroxyl, sulfhydryl, or halide. Representative polymermaleimides having bi- and tricyclic ring systems are provided in FIGS.1A and 1B.

The linkage, L, in structure I may further include a non-hydrocarbonportion adjacent to the polymer segment and interconnected to X asdescribed above. Exemplary non-hydrocarbon portions adjacent to thepolymer segment include —O—, —O—C(O)—NH—, —C(O)—NH—, —CH₂—C(O)—NH—,—NH—C(O)—O—, NH—C(O)—NH—, —NH—, and —S—, and are preferablyhydrolytically stable. Particularly preferred non-hydrocarbon portionsinclude —O—, —O—C(O)—NH—, —C(O)—NH—. In a less preferred embodiment, thenitrogen amide of the amide or carbamate function is a tertiarynitrogen, e.g., having a methyl or ethyl or similar group in place ofthe hydrogen as shown.

Exemplary linkages including a hydrocarbon chain according to thepresent invention are shown in Table 1 below.

TABLE 1 Exemplary linkers for Maleimide-Terminated Polymers

L₁ =

Designation P L1-AMDE ethylene, —(CH₂)₂— L1-AMPE pentamethylene,—(CH₂)₅— L1-MCH

L1-TEPE

L₂ = —NH—Q— Designation Q L2-TEME tetramethylene, —(CH₂)₄— L2-HEXAhexamethylene, —(CH₂)₆— L2-EPEN

L₃ = —O—Z Designation Z L3-ET ethylene, —(CH₂)₂— L3-TME trimethylene,—(CH₂)₃— L3-TEME tetramethylene, —(CH₂)₄— L3-PENT pentamethylene,—(CH₂)₅— L3-HEXA hexamethylene, —(CH₂)₆— L₄ = —CH₂—W— Designation WL4-PAHE —C(O)NH—(CH₂)₆— L4-BAHE —CH₂—C(O)NH—(CH₂)₆— L4-TMPA

L4-ETPA

L4-HEDA

L4-CMEN

  L₅ =

Designation V L5-TMPE

L5-HEXA —(CH₂)₆—

Many of the linkages in Table 1 are effective to retard hydrolysis ofthe maleimide ring, although some are more effective than others.Several linkages in Table 1 provide steric hindrance to attack of themaleimide ring nitrogen by water, making the maleimide resistant tohydrolysis. These linkers include those comprising L4-TMPA, L4-CMEN,L5-TMPE, L1-TEPE, L2-EPEN, L4-ETPA and L4-HEDA. Linkers comprisingL4-TMPA and L5-TMPE, which are based on the readily availablecorresponding symmetrical tertiary diamine, and L4-CMEN, which is basedon the commercially available diamine derivative of naturally occurringp-menthane, are exemplary linkages that reduce the rate of hydrolysis ofthe maleimide ring by providing both steric hindrance and adequatespacing between the ring and electron withdrawing groups. Particularlypreferred are linkers containing a cyclohexylene ring, as can be seenfrom the hydrolysis data provided in Table 4. As can be seen from thehydrolysis data, the 1,3-dimethylene-cyclohexylene linker imparts to theresultant PEG-maleimide a particular stability towards hydrolysis. Infact, its hydrolysis half life is 8 times longer than that of itslinkerless maleimide counterpart. The 1,4-dimethylene-cyclohexylenelinker also results in a stable maleimide polymer having a hydrolysishalf life that is over two and half times longer that that of itslinkerless maleimide counterpart.

The Polymer Segment

As shown in the illustrative structures above, a maleimide terminatedpolymer of the invention contains a water-soluble polymer segment.Representative POLYs include poly(alkylene glycols) such aspoly(ethylene glycol), poly(propylene glycol) (“PPG”), copolymers ofethylene glycol and propylene glycol, poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazoline, andpoly(N-acryloylmorpholine). POLY can be a homopolymer, an alternatingcopolymer, a random copolymer, a block copolymer, an alternatingtripolymer, a random tripolymer, or a block tripolymer of any of theabove. The water-soluble polymer segment is preferably, although notnecessarily, a poly(ethylene glycol) “PEG” or a derivative thereof.

The polymer segment can have any of a number of different geometries,for example, POLY can be linear, branched, or forked. Most typically,POLY is linear or is branched, for example, having 2 polymer arms.Although much of the discussion herein is focused upon PEG as anillustrative POLY, the discussion and structures presented herein can bereadily extended to encompass any of the water-soluble polymer segmentsdescribed above.

Any water-soluble polymer having at least one reactive terminus can beused to prepare a polymer maleimide in accordance with the invention andthe invention is not limited in this regard. Although water-solublepolymers bearing only a single reactive terminus can be used, polymersbearing two, three, four, five, six, seven, eight, nine, ten, eleven,twelve or more reactive termini suitable for conversion to a stabilizedpolymer maleimide as set forth herein can be used. Advantageously, asthe number of hydroxyl or other reactive moieties on the water-polymersegment increases, the number of available sites for introducing alinkered maleimido group increases. Nonlimiting examples of the upperlimit of the number of hydroxyl and/or reactive moieties associated withthe water-soluble polymer segment include from about 1 to about 500,from 1 to about 100, from about 1 to about 80, from about 1 to about 40,from about 1 to about 20, and from about 1 to about 10.

In turning now to the preferred POLY, PEG encompasses poly(ethyleneglycol) in any of its linear, branched or multi-arm forms, includingend-capped PEG, forked PEG, branched PEG, pendant PEG, and lesspreferably, PEG containing one or more degradable linkage separating themonomer subunits, to be more fully described below. In one embodiment ofthe invention, the polymer segment is absent an ester linkage.

A PEG polymer segment comprises the following: —(CH₂CH₂O)_(n)—CH₂CH₂—,where (n) typically ranges from about 3 to about 4,000, or from about 3to about 3,000, or more preferably from about 20 to about 1,000.

POLY can also be end-capped, for example an end-capped PEG where PEG isterminally capped with an inert end-capping group. Preferred end-cappedPEGs are those having as an end-capping moiety such as alkoxy,substituted alkoxy, alkenyloxy, substituted alkenyloxy, alkynyloxy,substituted alkynyloxy, aryloxy, substituted aryloxy. Preferredend-capping groups are methoxy, ethoxy, and benzyloxy. The end-cappinggroup can also advantageously comprise a phospholipid, although thepolymer may also be absent a lipid. Exemplary phospholipids includephosphatidylcholines, such as dilauroylphosphatidylcholine,dioleylphosphatidylcholine, dipalmitoylphosphatidylcholine,disteroylphosphatidylcholine, behenoylphosphatidylcholine,arachidoylphosphatidylcholine, and lecithin.

Referring now to any of the structures containing a polymer segment,POLY, POLY may correspond or comprise the following:“Z—(CH₂CH₂O)_(n)—” or “Z—(CH₂CH₂O)_(n)—CH₂CH₂—”,

where n ranges from about 3 to about 4000, or from about 10 to about4000, and Z is or includes a functional group, which may be a reactivegroup or an end-capping group. Examples of Z include hydroxy, amino,ester, carbonate, aldehyde, acetal, aldehyde hydrate, ketone, ketal,ketone hydrate, alkenyl, acrylate, methacrylate, acrylamide, sulfone,thiol, carboxylic acid, isocyanate, isothiocyanate, hydrazide, urea,maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide,alkoxy, benzyloxy, silane, lipid, phospholipid, biotin, and fluorescein,including activated and protected forms thereof where applicable.Preferred are functional groups such as N-hydroxysuccinimidyl ester,benzotriazolyl carbonate, amine, vinylsulfone, maleimide, N-succinimidylcarbonate, hydrazide, succinimidyl propionate, succinimidyl butanoate,succinimidyl succinate, succinimidyl ester, glycidyl ether,oxycarbonylimidazole, p-nitrophenyl carbonate, aldehyde,orthopyridyl-disulfide, and acrylol.

These and other functional groups, Z, are described in the followingreferences, all of which are incorporated by reference herein:N-succinimidyl carbonate (see e.g., U.S. Pat. Nos. 5,281,698,5,468,478), amine (see, e.g., Buckmann et al. Makromol. Chem. 182:1379(1981), Zalipsky et al. Eur. Polym. J. 19:1177 (1983)), hydrazide (See,e.g., Andresz et al. Makromol. Chem. 179:301 (1978)), succinimidylpropionate and succinimidyl butanoate (see, e.g., Olson et al. inPoly(ethylene glycol) Chemistry & Biological Applications, pp 170-181,Harris & Zalipsky Eds., ACS, Washington, D.C., 1997; see also U.S. Pat.No. 5,672,662), succinimidyl succinate (See, e.g., Abuchowski et al.Cancer Biochem. Biophys. 7:175 (1984) and Joppich et al., Makromol.Chem. 180:1381 (1979), succinimidyl ester (see, e.g., U.S. Pat. No.4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat. No.5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J. Biochem.94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354 (1991),oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal. Biochem.131:25 (1983), Tondelli et al. J. Controlled Release 1:251 (1985)),p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl. Biochem.Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem. Biotech.,27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym. Sci. Chem.Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No. 5,252,714),maleimide (see, e.g., Goodson et al. Bio/Technology 8:343 (1990), Romaniet al. in Chemistry of Peptides and Proteins 2:29 (1984)), and Kogan,Synthetic Comm. 22:2417 (1992)), orthopyridyl-disulfide (see, e.g.,Woghiren, et al. Bioconj. Chem. 4:314 (1993)), acrylol (see, e.g.,Sawhney et al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g.,U.S. Pat. No. 5,900,461).

Again, the POLY structures shown immediately above may represent linearpolymer segments, or may form part of a branched or forked polymersegment. In an instance where the polymer segment is branched, the POLYstructures immediately above may, for example, correspond to the polymerarms forming part of the overall POLY structure. Alternatively, in aninstance where POLY possesses a forked structure, the above POLYstructure may, for example, correspond to the linear portion of thepolymer segment prior to the branch point.

POLY may also correspond to a branched PEG molecule having 2 arms, 3arms, 4 arms, 5 arms, 6 arms, 7 arms, 8 arms or more. Branched polymersused to prepare the polymer maleimides of the invention may possessanywhere from 2 to 300 or so reactive termini. Preferred are branchedpolymer segments having 2 or 3 polymer arms. An illustrative branchedPOLY, as described in U.S. Pat. No. 5,932,462, corresponds to thestructure:

In this representation, R″ is a nonreactive moiety, such as H, methyl ora PEG, and P and Q are nonreactive linkages. In a preferred embodiment,the branched PEG polymer segment is methoxy poly(ethylene glycol)disubstituted lysine.

In the above particular branched configuration, the branched polymersegment possesses a single reactive site extending from the “C” branchpoint for positioning of the reactive maleimide group via a linker asdescribed herein. Branched PEGs such as these for use in the presentinvention will typically have fewer than 4 PEG arms, and morepreferably, will have 2 or 3 PEG arms. Such branched PEGs offer theadvantage of having a single reactive site, coupled with a larger, moredense polymer cloud than their linear PEG counterparts.

One particular type of branched PEG maleimide corresponds to thestructure: (MeO-PEG-)_(i)G-[O]_(b)—C(O)—NH—X-MAL, where MAL representsmaleimide, i equals 2 or 3, and G is a lysine or other suitable aminoacid residue.

An illustrative branched polymer maleimide of the invention has thestructure shown below, where X is any of the herein describedhydrolytically stable linkers.

The synthesis of a polymer of the invention having the structuralfeatures embodied in XVIII above is provided in Example 1. Branched PEGsfor use in preparing a polymer maleimide of the invention additionallyinclude those represented more generally by the formula R(PEG)_(n),where R is a central or core molecule from which extends 2 or more PEGarms. The variable n represents the number of PEG arms, where each ofthe polymer arms can independently be end-capped or alternatively,possess a reactive functional group at its terminus, such as a maleimideor other reactive functional group. In such multi-armed embodiments ofthe invention, each PEG arm typically possesses a maleimide group at itsterminus. Branched PEGs such as those represented generally by theformula, R(PEG)_(d), above possess 2 polymer arms to about 300 polymerarms (i.e., n ranges from 2 to about 300). Branched PEGs such as thesepreferably possess from 2 to about 25 polymer arms, more preferably from2 to about 20 polymer arms, and even more preferably from 2 to about 15polymer arms or fewer. Most preferred are multi-armed polymers having 3,4, 5, 6, 7 or 8 arms.

Preferred core molecules in branched PEGs as described above arepolyols. Such polyols include aliphatic polyols having from 1 to 10carbon atoms and from 1 to 10 hydroxyl groups, including ethyleneglycol, alkane diols, alkyl glycols, alkylidene alkyl diols, alkylcycloalkane diols, 1,5-decalindiol,4,8-bis(hydroxymethyl)tricyclodecane, cycloalkylidene diols,dihydroxyalkanes, trihydroxyalkanes, and the like. Cycloaliphaticpolyols may also be employed, including straight chained or closed-ringsugars and sugar alcohols, such as mannitol, sorbitol, inositol,xylitol, quebrachitol, threitol, arabitol, erythritol, adonitol,dulcitol, facose, ribose, arabinose, xylose, lyxose, rhamnose,galactose, glucose, fructose, sorbose, mannose, pyranose, altrose,talose, tagitose, pyranosides, sucrose, lactose, maltose, and the like.Additional aliphatic polyols include derivatives of glyceraldehyde,glucose, ribose, mannose, galactose, and related stereoisomers. Othercore polyols that may be used include crown ether, cyclodextrins,dextrins and other carbohydrates such as starches and amylose. Preferredpolyols include glycerol, pentaerythritol, sorbitol, andtrimethylolpropane.

A representative multi-arm polymer structure of the type described aboveis:

where d is an integer from 3 to about 100, and R is a residue of acentral core molecule having 3 or more hydroxyl groups, amino groups, orcombinations thereof.

Multi-armed PEGs for use in preparing a polymer maleimide of theinvention include multi-arm PEGs available from Nektar, Huntsville, Ala.In a preferred embodiment, a multi-armed polymer maleimide of theinvention corresponds to the following, where the specifics of thelinkered maleimide portion of the molecule are provided elsewhereherein.

-   -   where        -   PEG is —(CH₂CH₂O)_(n)CH₂CH₂—,        -   M is:

and m is selected from the group consisting of 3, 4, 5, 6, 7, and 8.

Alternatively, the polymer maleimide may possess an overall forkedstructure. An example of a forked PEG corresponds to the structure:

where PEG is any of the forms of PEG described herein, A is a linkinggroup, preferably a hydrolytically stable linkage such as oxygen,sulfur, or —C(O)—NH—, F and F′ are hydrolytically stable spacer groupsthat are optionally present, and the other variables corresponding tothe hydrolytically stable linker, X, and maleimide (MAL) portion are asdefined above. Both the general and specific descriptions of possiblevalues for X are applicable to the embodiment above, structure XVIII.Examplary linkers and spacer groups corresponding to A, F and F′ aredescribed in International Application No. PCT/US99/05333, and areuseful in forming polymer segments of this type for use in the presentinvention. F and F′ are spacer groups that may be the same of different.In one particular embodiment of the above, PEG is mPEG, A corresponds to—C(O)—NH—, and F and F′ are both methylene or —CH₂—. This type ofpolymer segment is useful for reaction with two active agents, where thetwo active agents are positioned a precise or predetermined distanceapart, depending upon the selection of F and F′.

Another version of a polymer reagent of the invention having a forkedpolymer segment corresponds to:

where the variables are as described above. Preferably, X in thisembodiment is a saturated cyclic or alicyclic hydrocarbon chain having atotal of 3 to about 20 carbon atoms.

An exemplary branched PEG corresponding to “PEG” in the above formula ismPEG disubstituted lysine, where “PEG” corresponds to:

Alternatively, the PEG polymer segment for use in preparing a polymermaleimide of the invention may be a PEG molecule having pendant reactivegroups along the length of the PEG chain rather than at the end(s), toyield a stabilized polymer maleimide having one or more pendantmaleimide groups attached to the PEG chain by a linker, X.

Further, in a less preferred embodiment, the polymer segment itself maypossess one or more weak or degradable linkages that are subject tohydrolysis. Illustrative degradable linkages that may be present in thepolymer segment include but are not limited to carbonate, imine,phosphate ester, and hydrazone.

Generally, the nominal average molecular mass of the water-solublepolymer segment, POLY will vary. The nominal average molecular mass ofPOLY typically falls in one or more of the following ranges: about 100daltons to about 100,000 daltons; from about 500 daltons to about 80,000daltons; from about 1,000 daltons to about 50,000 daltons; from about2,000 daltons to about 25,000 daltons; from about 5,000 daltons to about20,000 daltons. Exemplary nominal average molecular masses for thewater-soluble polymer segment POLY include about 1,000 daltons, about5,000 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000daltons, about 25,000 daltons, about 30,000 daltons, and about 40,000daltons. Low molecular weight POLYs possess molecular masses of about250, 500, 750, 1000, 2000, or 5000 daltons.

Polymer Amines

The present invention also extends to amine counterparts of any and allof the above-described structures, with the exception that theheretofore described maleimide ring is replaced with an amino group,preferably —NH₂.

More particularly, the invention extends to a water-soluble polymerhaving the structure:

where POLY, b and X are as described above.

Representative polymer amines include those having the generalizedstructures shown below:

wherein q and p each independently range from 0 to 6, and thesubstituents on said cyclohexylene ring are either cis or trans.

wherein q and p each independently range from 0 to 6, and thesubstituents on said cyclohexylene ring are either cis or trans.

The amine-terminated polymers represented generally by VIII have anumber of uses to be described in greater detail below. For instance,they can be converted to the corresponding maleimide-terminated polymersof the invention, or alternatively, used without further modificationfor covalent attachment to active agents or surfaces, or for forminghydrogels.

Hyrolytic Stability and Methods of Preparation

As described above, the polymer maleimides provided herein are resistantto hydrolysis, as demonstrated for both the polymer reagents themselves(Examples VII and XIII) and for their corresponding conjugates (ExampleVIII). A polymer maleimide of the invention has a hydrolysis half-lifethat is longer than that of its linkerless polymer maleimidecounterpart, when measured under the same conditions. That is to say, aTinkered polymer maleimide of the invention possesses a rate ofhydrolysis that is slower than that of its corresponding linkerlessversion, meaning that the maleimide ring remains intact longer underessentially the same conditions. For instance, in looking at thehydrolysis data in Examples 7 and 14, Tables 2 and 4, each of therepresentative linkers has a hydrolysis half-life that is extended overthat of the linkerless maleimide (“3-ET”).

The polymer maleimides of the invention can be prepared by a number ofalternative routes including the following. In one approach, amaleimide-terminated polymer of the invention is prepared by reacting afunctional group attached to a polymer segment (i.e., an activatedpolymer segment) with a functional group attached to a bifunctionallinker. Reacting the polymer segment with a bifunctional linker reagentresults in covalent attachment, through a hydrolytically stable linkage,of the linker to the polymer segment. The remaining functional group onthe bifunctional linker reagent is either a maleimide or a functionalgroup that can be readily converted to a maleimide.

For example, the linker reagent may possess the structure A-L-B, whereinA is a first functional group that is reactive with a second functionalgroup on the polymer segment to thereby form a hydrolytically stablelinkage, L, to form POLY-L-B, where B is a maleimide or a functionalgroup that can be readily converted to a maleimide (e.g., an amine thatcan be converted to a maleimide by reaction withmethoxycarbonylmaleimide). In the above approach, A can be any of anumber of functional groups such as halo, hydroxyl, active ester such asN-succinimidyl ester, active carbonate, acetal, aldehyde, aldehydehydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone,thiol, carboxylic acid, isocyanate, isothiocyanate, maleimide,vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, and epoxide.Particular examples of linker reagents include 1,4-dibromobutane,1,5-dibromopentane, 1,6-dibromohexane, N-succinimidylε-maleimidohexanoate), N-succinimidyl(ε-maleimidopentanoate),N-(γ-maleimidobutryloxy)succinimide ester,N-(γ-maleimidocaproyloxy)succinimide ester,4,7,10-trioxa-1,3-tridecanediamine,4-(maleimidomethyl)-1-cyclohexanecarboxylic acid—NHS ester,tetramethylenediamine, pentamethylenediamine, hexamethylenediamine,2,5-diamino-2,5-dimethylhexane, 1,3-cyclohexylbis(methylamine), and1,4-cyclohexylbis(methylamine). Such linker reagents are eithercommercially available, for example from Pierce Chemical Company, or canbe prepared from commercially available starting materials usingmethodology known in the art.

This approach is shown more particularly as method 1 below, as itrelates to the formation of polymer malemides containing an amide orurethane bond connecting the polymer segment to the linker, X.

Method 1.

In a method similar to the above, designated method 2, the reactivepolymer starting material, POLY-[O]_(b)—C(O)-LG, is reacted with adiamine reagent, H₂N—X—NH₂, to form the corresponding polymer amineintermediate, POLY-[O]_(b)—C(O)—HN—X—NH. This intermediate is thenconverted to the corresponding stabilized maleimide-terminated polymer.This method is advantageous in that many diamine reagents suitable forforming a stabilized polymer maleimide as described herein arecommercially available. Moreover, polymer-amine intermediates can bemore easily purified than their maleimide counterparts, e.g., by columnchromatography, to thereby provide a polymer maleimide product that issignificantly absent other undesirable polymer-derived side-products,such as PEG-diol and PEG-diol derived impurities. Method 2 is shownbelow.

Method 2.

In methods 1 and 2, LG represents a leaving group, while the othervariables are as described previously. The reactive polymer startingmaterial, POLY-[O]_(b)—C(O)-LG, may be, for example, an acyl halide, ahaloformate, an anhydride, or an active ester. Leaving groups useful inthe methods include halides (e.g., chloro, bromo, and iodo),N-hydroxysuccinimide, N-hydroxybenzotriazole, and para-nitrophenolate.The coupling reaction to form the amide or urethane bond is generallycarried out in a dry organic solvent, preferably under an inertatmosphere such as nitrogen or argon. Suitable solvents includeacetonitrile, chlorinated hydrocarbons such as chloroform anddichloromethane, aromatic hydrocarbons such as benzene, toluene, andxylene, and solvents such as acetone, and tetrahydrofuran. The reactionis typically carried out at temperatures ranging from about 0 to 100°C., depending upon the type of solvent employed and the reactivity ofthe particular reagents themselves. The coupling is generally conductedin the presence of a base. Bases include trialkylamines such as triethylamine, pyridine, 4-(dimethylamino)pyridine, and inorganic bases such assodium carbonate.

Representative bicyclic and tricyclic diamine reactants corresponding toH₂N—X—NH₂ in method 2 above are provided in FIG. 2. In method 2, it maybe necessary in certain instances to protect one of the amino groups inH₂N—X—NH₂ using, for example, a conventional amino-protecting group suchas t-BOC or FMOC. The protecting group in POLY-[O]_(b)—C(O)—HN—X—NH isthen typically removed prior to further purification or transformation.See for example, Examples 10 and 11. In instances where purification ofthe intermediate polymer amine is undertaken, any of a number ofpurification approaches can be used such as precipitation orchromatography, although preferred is ion exchange chromatography due tothe presence of an amino group on the intermediate polymer amine.

In continuing with the approach in method 2, the intermediate polymeramine is then converted to the corresponding maleimide. Generally, thisconversion is carried out by reacting POLY-[O]_(b)—C(O)—H₂N—X—NH₂ with areagent such as N-methoxycarbonylmaleimide,exo-7-oxa[2.2.1]bicycloheptane-2,3-dicarboxylic anhydride, or maleicanhydride, under conditions suitable for formingPOLY-[O]_(b)—C(O)—H₂N—X-MAL.

A preferred reagent is N-methoxycarbonylmaleimide, and in this instance,the conversion to the maleimide is carried out in water or an aqueousmixture of water and a water miscible solvent such as acetonitrile oracetone. The conversion reaction is generally carried out attemperatures ranging from about 0 to 80° C., at pHs ranging from about6.5 to 9

When the reagent is maleic anhydride, POLY-[O]_(b)—C(O)—H₂N—X—NH₂ isreacted with maleic anhydride under conditions effective to formPOLY-[O]_(b)—C(O)—NH—X—NH—C(O)CH═CHCOOH (XI) as an intermediate. Thisintermediate is then heated under conditions effective to promotecyclization by elimination of water to form POLY-[O]_(b)—C(O)—NH—X-MAL.The efficiency of the cyclization reaction to form the maleimide ringtypically ranges from about 15 to about 80 percent.

Generally, the product, POLY-[O]_(b)—C(O)—H₂N—X-MAL is recovered fromthe reaction mixture, and optionally further purified. In instanceswhere the product is formed by method 2, further purification, forexample, to remove polymer-derived impurities, may be unnecessary ifpurification is carried out on the amine-precursor, for example, by ionexchange chromatography. Preferably, the recovered product,POLY-[O]_(b)—C(O)—H₂N—X-MAL has a polymer purity of greater than about80%.

Examples 1 and 5 illustrate a method of forming a reactive polymer ofthe invention using a linker that comprises a terminal maleimide group.In Example 1, a polymer segment modified to contain a reactive aminogroup is reacted with bifunctional linker comprising an activated esterand a maleimide group. A similar approach is utilized in Example 5 toprepare a polymer maleimide having a cyclohexylene containing linker.Examples 2, 3, and 4 demonstrate formation of a polymer amineintermediate that is then converted to the corresponding maleimide.Examples 9, 10, 11, and 12 demonstrate the synthesis ofcycloalkylene-containing linkers by method 2 above, where a reactivepolymer starting material, POLY-[O]_(b)—C(O)-LG, is reacted with adiamine reagent to form POLY-[O]_(b)—C(O)—H₂N—X—NH₂, which is thenconverted to the corresponding maleimide-terminated product.

Storage of Polymer Maleimide Reagents

Preferably, the polymer maleimides of the invention, as well as theiramino counterparts, are stored under an inert atmosphere, such as underargon or under nitrogen. Due to the potential of the maleimide portionof the molecule for reaction with water (e.g., by exposure to moistureto form the corresponding ring-opened form), it is also preferable tominimize exposure of the polymer maleimides of the invention tomoisture. Thus, preferred storage conditions are under dry argon oranother dry inert gas at temperatures below about −15° C. Storage underlow temperature conditions is preferred, since rates of undesirable sidereactions, such as maleimide ring opening, are slowed at lowertemperatures. In instances where the polymer segment of the polymerproduct is PEG, the PEG portion can react slowly with oxygen to formperoxides along the PEG portion of the molecule. Formation of peroxidescan ultimately lead to chain cleavage, thus increasing thepolydispersity of the PEG reagents provided herein. In view of theabove, it is additionally preferred to store the PEG maleimides andrelated polymers of the invention in the dark.

Biologically Active Conjugates

Coupling Chemistry, Separation, Storage

The Conjugates

The present invention also encompasses conjugates formed by reaction ofany of the herein described stabilized polymer maleimides or theircorresponding polymer amine counterparts. In particular, theherein-described polymer maleimides are useful for conjugation to activeagents or surfaces bearing at least one thiol or amino group availablefor reaction, while the herein-described polymer amines are useful forconjugation to active agents or surfaces bearing at least one carboxylicgroup available for reaction.

For instance, a conjugate of the invention may possess the followingstructure:

where “—S-active agent” represents an active agent, preferably abiologically active agent, comprising a thiol (—SH) group, and the othervariables are as described previously. In instances where the activeagent is a biologically active agent or small molecule containing onlyone reactive thiol group, the resulting composition may advantageouslycontain only a single polymer conjugate species, due to the relativelylow number of sulfhydryl groups typically contained within a protein andaccessible for conjugation. In some instances, a protein or smallmolecule or other active agent is engineered to possess a thiol group ina known position, and will similarly result in a composition comprisingonly a single polymer conjugate species.

Alternatively, a conjugate of the invention may possess the followingstructure:

In structure XVI, “—NH-active agent” represents an active agent orsurface comprising an amino group, preferably a biologically activeagent, and the other variables are as previously described.

The polymer amines of the invention, when used directly, can be used toprovide conjugates of the following type:

The polymer conjugates provided herein, particularly those derived fromthe stabilized polymer maleimides of the invention, similarly possessthe feature of improved hydrolytic stability to maleimide ring opening.This feature is demonstrated in Example VIII. The synthesis of exemplaryconjugates, using both the model compound, 2-mercaptoethanol, and anillustrative protein, is described in Examples 6, 14, 15, 16, and 17.

Methods of Conjugation

Suitable conjugation conditions are those conditions of time,temperature, pH, reagent concentration, solvent, and the like sufficientto effect conjugation between a polymeric reagent and an active agent.As is known in the art, the specific conditions depend upon, among otherthings, the active agent, the type of conjugation desired, the presenceof other materials in the reaction mixture and so forth. Sufficientconditions for effecting conjugation in any particular case can bedetermined by one of ordinary skill in the art upon a reading of thedisclosure herein, reference to the relevant literature, and/or throughroutine experimentation.

Exemplary conjugation conditions include carrying out the conjugationreaction at a pH of from about 6 to about 10, and at, for example, a pHof about 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10. The reaction isallowed to proceed from about 5 minutes to about 72 hours, preferablyfrom about 30 minutes to about 48 hours, and more preferably from about4 hours to about 24 hours or less. Temperatures for conjugationreactions are typically, although not necessarily, in the range of fromabout 0° C. to about 40° C.; conjugation is often carried out at roomtemperature or less. Conjugation reactions are often carried out in abuffer such as a phosphate or acetate buffer or similar system.

With respect to reagent concentration, an excess of the polymericreagent is typically combined with the active agent. In some cases,however, it is preferred to have stoichiometic amounts of the number ofreactive groups on the polymeric reagent to the amount of active agent.Exemplary ratios of polymeric reagent to active agent include molarratios of about 1:1 (polymeric reagent:active agent), 1.5:1, 2:1, 3:1,4:1, 5:1, 6:1, 8:1, or 10:1. The conjugation reaction is allowed toproceed until substantially no further conjugation occurs, which cangenerally be determined by monitoring the progress of the reaction overtime.

Progress of the reaction can be monitored by withdrawing aliquots fromthe reaction mixture at various time points and analyzing the reactionmixture by SDS-PAGE or MALDI-TOF mass spectrometry or any other suitableanalytical method. Once a plateau is reached with respect to the amountof conjugate formed or the amount of unconjugated polymer remaining, thereaction is assumed to be complete. Typically, the conjugation reactiontakes anywhere from minutes to several hours (e.g., from 5 minutes to 24hours or more). The resulting product mixture is preferably, but notnecessarily purified, to separate out excess reagents, unconjugatedreactants (e.g., active agent) undesired multi-conjugated species, andfree or unreacted polymer. The resulting conjugates can then be furthercharacterized using analytical methods such as MALDI, capillaryelectrophoresis, gel electrophoresis, and/or chromatography.

More preferably, a polymer maleimide of the invention is typicallyconjugated to a sulfhydryl-containing active agent at pHs ranging fromabout 6-9 (e.g., at 6, 6.5, 7, 7.5, 8, 8.5, or 9), more preferably atpHs from about 7-9, and even more preferably at pHs from about 7 to 8.Generally, a slight molar excess of polymer maleimide is employed, forexample, a 1.5 to 15-fold molar excess, preferably a 2-fold to 10 foldmolar excess. Reaction times generally range from about 15 minutes toseveral hours, e.g., 8 or more hours, at room temperature. Forsterically hindered sulfhydryl groups, required reaction times may besignificantly longer. The stabilized maleimides of the invention arethiol-selective, and thiol-selective conjugation is preferably conductedat pHs around 7.

Reactions with amino groups proceed at higher pHs, but are relativelyslow. Protein PEGylation reaction conditions vary depending on theprotein, the desired degree of PEGylation, and the particular polymermaleimide reagent.

Separation

Optionally, conjugates produced by reacting a PEG maleimide or PEG amineof the invention with a biologically active agent are purified toobtain/isolate different PEGylated species. Alternatively, and morepreferably for lower molecular weight PEGs, e.g., having molecularweights less than about 20 kilodaltons, preferably less than or equal toabout 10 kilodaltons, a product mixture can be purified to obtain adistribution around a certain number of PEGs per protein molecule, whereapplicable. For example, a product mixture can be purified to obtain anaverage of anywhere from one to five PEGs per protein, typically anaverage of about 3 PEGs per protein. The strategy for purification ofthe final conjugate reaction mixture will depend upon a number offactors—the molecular weight of the polymer employed, the particularprotein, the desired dosing regimen, and the residual activity and invivo properties of the individual conjugate(s) species.

If desired, PEG conjugates having different molecular weights can beisolated using gel filtration chromatography. While this approach can beused to separate PEG conjugates having different molecular weights, thisapproach is generally ineffective for separating positional isomershaving different pegylation sites within a protein. For example, gelfiltration chromatography can be used to separate from each othermixtures of PEG 1-mers, 2-mers, 3-mers, etc., although each of therecovered PEG-mer compositions may contain PEGs attached to differentreactive amino groups (e.g., lysine residues) within the protein.

Gel filtration columns suitable for carrying out this type of separationinclude Superdex™ and Sephadex™ columns available from AmershamBiosciences. Selection of a particular column will depend upon thedesired fractionation range desired. Elution is generally carried outusing a non-amine based buffer, such as phosphate, acetate, or the like.The collected fractions may be analysed by a number of differentmethods, for example, (i) OD at 280 nm for protein content, (ii) BSAprotein analysis, (iii) iodine testing for PEG content (Sims G. E. C.,et al., Anal. Biochem, 107, 60-63, 1980), or alternatively, (iv) byrunning an SDS PAGE gel, followed by staining with barium iodide.

Separation of positional isomers is carried out by reverse phasechromatography using an RP-HPLC C18 column (Amersham Biosciences orVydac) or by ion exchange chromatography using an ion exchange column,e.g., a Sepharose™ ion exchange column available from AmershamBiosciences. Either approach can be used to separate PEG-biomoleculeisomers having the same molecular weight (positional isomers).

Depending upon the intended use for the resulting PEG-conjugates,following conjugation, and optionally additional separation steps, theconjugate mixture may be concentrated, sterile filtered, and stored atlow temperatures from about −20° C. to about −80° C. Alternatively, theconjugate may be lyophilized, either with or without residual buffer andstored as a lyophilized powder. In some instances, it is preferable toexchange a buffer used for conjugation, such as sodium acetate, for avolatile buffer such as ammonium carbonate or ammonium acetate, that canbe readily removed during lyophilization, so that the lyophilizedprotein conjugate powder formulation is absent residual buffer.Alternatively, a buffer exchange step may be used using a formulationbuffer, so that the lyophilized conjugate is in a form suitable forreconstitution into a formulation buffer and ultimately foradministration to a mammal.

Target Molecules and Surfaces

The stabilized polymer maleimides (amines) of the invention may beattached, either covalently or non-covalently, to a number of entitiesincluding films, chemical separation and purification surfaces, solidsupports, metal/metal oxide surfaces such as gold, titanium, tantalum,niobium, aluminum, steel, and their oxides, silicon oxide,macromolecules, and small molecules. Additionally, the polymers of theinvention may also be used in biochemical sensors, bioelectronicswitches, and gates. The polymer maleimides (amines) of the inventionmay also be employed as carriers for peptide synthesis, for thepreparation of polymer-coated surfaces and polymer grafts, to preparepolymer-ligand conjugates for affinity partitioning, to preparecross-linked or non-cross-linked hydrogels, and to preparepolymer-cofactor adducts for bioreactors.

A biologically active agent for use in coupling to a polymer of theinvention may be any one or more of the following. Suitable agents maybe selected from, for example, hypnotics and sedatives, psychicenergizers, tranquilizers, respiratory drugs, anticonvulsants, musclerelaxants, antiparkinson agents (dopamine antagnonists), analgesics,anti-inflammatories, antianxiety drugs (anxiolytics), appetitesuppressants, antimigraine agents, muscle contractants, anti-infectives(antibiotics, antivirals, antifungals, vaccines) antiarthritics,antimalarials, antiemetics, anepileptics, bronchodilators, cytokines,growth factors, anti-cancer agents, antithrombotic agents,antihypertensives, cardiovascular drugs, antiarrhythmics, antioxicants,anti-asthma agents, hormonal agents including contraceptives,sympathomimetics, diuretics, lipid regulating agents, antiandrogenicagents, antiparasitics, anticoagulants, neoplastics, antineoplastics,hypoglycemics, nutritional agents and supplements, growth supplements,antienteritis agents, vaccines, antibodies, diagnostic agents, andcontrasting agents.

More particularly, the active agent may fall into one of a number ofstructural classes, including but not limited to small molecules(preferably insoluble small molecules), peptides, polypeptides,proteins, antibodies, polysaccharides, steroids, nucleotides,oligonucleotides, polynucleotides, fats, electrolytes, and the like.Preferably, an active agent for coupling to a polymer maleimide of theinvention possesses a native amino or a sulfydryl group, oralternatively, is modified to contain at least one reactive amino orsulfhydryl group suitable for coupling to a polymer maleimide of theinvention.

Specific examples of active agents suitable for covalent attachment to apolymer of the invention include but are not limited to aspariginase,amdoxovir (DAPD), antide, becaplermin, calcitonins, cyanovirin,denileukin diftitox, erythropoietin (EPO), EPO agonists (e.g., peptidesfrom about 10-40 amino acids in length and comprising a particular coresequence as described in WO 96/40749), dornase alpha, erythropoiesisstimulating protein (NESP), coagulation factors such as Factor V, FactorVII, Factor VIIa, Factor VIII, Factor IX, Factor X, Factor XII, FactorXIII, von Willebrand factor; ceredase, cerezyme, alpha-glucosidase,collagen, cyclosporin, alpha defensins, beta defensins, exedin-4,granulocyte colony stimulating factor (GCSF), thrombopoietin (TPO),alpha-1 proteinase inhibitor, elcatonin, granulocyte macrophage colonystimulating factor (GMCSF), fibrinogen, filgrastim, growth hormoneshuman growth hormone (hGH), growth hormone releasing hormone (GHRH),GRO-beta, GRO-beta antibody, bone morphogenic proteins such as bonemorphogenic protein-2, bone morphogenic protein-6, OP-1; acidicfibroblast growth factor, basic fibroblast growth factor, CD-40 ligand,heparin, human serum albumin, low molecular weight heparin (LMWH),interferons such as interferon alpha, interferon beta, interferon gamma,interferon omega, interferon tau, consensus interferon; interleukins andinterleukin receptors such as interleukin-1 receptor, interleukin-2,interluekin-2 fusion proteins, interleukin-1 receptor antagonist,interleukin-3, interleukin-4, interleukin-4 receptor, interleukin-6,interleukin-8, interleukin-12, interleukin-13 receptor, interleukin-17receptor; lactoferrin and lactoferrin fragments, luteinizing hormonereleasing hormone (LHRH), insulin, pro-insulin, insulin analogues (e.g.,mono-acylated insulin as described in U.S. Pat. No. 5,922,675), amylin,C-peptide, somatostatin, somatostatin analogs including octreotide,vasopressin, follicle stimulating hormone (FSH), influenza vaccine,insulin-like growth factor (IGF), insulintropin, macrophage colonystimulating factor (M-CSF), plasminogen activators such as alteplase,urokinase, reteplase, streptokinase, pamiteplase, lanoteplase, andteneteplase; nerve growth factor (NGF), osteoprotegerin,platelet-derived growth factor, tissue growth factors, transforminggrowth factor-1, vascular endothelial growth factor, leukemia inhibitingfactor, keratinocyte growth factor (KGF), glial growth factor (GGF), TCell receptors, CD molecules/antigens, tumor necrosis factor (TNF),monocyte chemoattractant protein-1, endothelial growth factors,parathyroid hormone (PTH), glucagon-like peptide, somatotropin, thymosinalpha 1, thymosin alpha 1 IIb/IIIa inhibitor, thymosin beta 10, thymosinbeta 9, thymosin beta 4, alpha-1 antitrypsin, phosphodiesterase (PDE)compounds, VLA-4 (very late antigen-4), VLA-4 inhibitors,bisphosponates, respiratory syncytial virus antibody, cystic fibrosistransmembrane regulator (CFTR) gene, deoxyreibonuclease (Dnase),bactericidal/permeability increasing protein (BPI), and anti-CMVantibody. Exemplary monoclonal antibodies include etanercept (a dimericfusion protein consisting of the extracellular ligand-binding portion ofthe human 75 kD TNF receptor linked to the Fc portion of IgG1),abciximab, afeliomomab, basiliximab, daclizumab, infliximab, ibritumomabtiuexetan, mitumomab, muromonab-CD3, iodine 131 tositumomab conjugate,olizumab, rituximab, and trastuzumab (herceptin).

Additional agents suitable for covalent attachment to a polymer of theinvention include but are not limited to amifostine, amiodarone,aminocaproic acid, aminohippurate sodium, aminoglutethimide,aminolevulinic acid, aminosalicylic acid, amsacrine, anagrelide,anastrozole, asparaginase, anthracyclines, bexarotene, bicalutamide,bleomycin, buserelin, busulfan, cabergoline, capecitabine, carboplatin,carmustine, chlorambucin, cilastatin sodium, cisplatin, cladribine,clodronate, cyclophosphamide, cyproterone, cytarabine, camptothecins,13-cis retinoic acid, all trans retinoic acid; dacarbazine,dactinomycin, daunorubicin, deferoxamine, dexamethasone, diclofenac,diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estramustine,etoposide, exemestane, fexofenadine, fludarabine, fludrocortisone,fluorouracil, fluoxymesterone, flutamide, gemcitabine, epinephrine,L-Dopa, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan,itraconazole, goserelin, letrozole, leucovorin, levamisole, lisinopril,lovothyroxine sodium, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, metaraminol bitartrate,methotrexate, metoclopramide, mexiletine, mitomycin, mitotane,mitoxantrone, naloxone, nicotine, nilutamide, octreotide, oxaliplatin,pamidronate, pentostatin, pilcamycin, porfimer, prednisone,procarbazine, prochlorperazine, ondansetron, raltitrexed, sirolimus,streptozocin, tacrolimus, tamoxifen, temozolomide, teniposide,testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa,topotecan, tretinoin, valrubicin, vinblastine, vincristine, vindesine,vinorelbine, dolasetron, granisetron; formoterol, fluticasone,leuprolide, midazolam, alprazolam, amphotericin B, podophylotoxins,nucleoside antivirals, aroyl hydrazones, sumatriptan; macrolides such aserythromycin, oleandomycin, troleandomycin, roxithromycin,clarithromycin, davercin, azithromycin, flurithromycin, dirithromycin,josamycin, spiromycin, midecamycin, leucomycin, miocamycin, rokitamycin,andazithromycin, and swinolide A; fluoroquinolones such asciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin,moxifloxicin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin,lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin,fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin,clinafloxacin, and sitafloxacin; aminoglycosides such as gentamicin,netilmicin, paramecin, tobramycin, amikacin, kanamycin, neomycin, andstreptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin,daptomycin, gramicidin, colistimethate; polymixins such as polymixin B,capreomycin, bacitracin, penems; penicillins includingpenicllinase-sensitive agents like penicillin G, penicillin V;penicllinase-resistant agents like methicillin, oxacillin, cloxacillin,dicloxacillin, floxacillin, nafcillin; gram negative microorganismactive agents like ampicillin, amoxicillin, and hetacillin, cillin, andgalampicillin; antipseudomonal penicillins like carbenicillin,ticarcillin, azlocillin, mezlocillin, and piperacillin; cephalosporinslike cefpodoxime, cefprozil, ceftbuten, ceftizoxime, ceftriaxone,cephalothin, cephapirin, cephalexin, cephradrine, cefoxitin,cefamandole, cefazolin, cephaloridine, cefaclor, cefadroxil,cephaloglycin, cefuroxime, ceforanide, cefotaxime, cefatrizine,cephacetrile, cefepime, cefixime, cefonicid, cefoperazone, cefotetan,cefinetazole, ceftazidime, loracarbef, and moxalactam, monobactams likeaztreonam; and carbapenems such as imipenem, meropenem, pentamidineisethiouate, albuterol sulfate, lidocaine, metaproterenol sulfate,beclomethasone diprepionate, triamcinolone acetamide, budesonideacetonide, fluticasone, ipratropium bromide, flunisolide, cromolynsodium, and ergotamine tartrate; taxanes such as paclitaxel; SN-38, andtyrphostines.

Preferred peptides or proteins for coupling to a polymer maleimide ofthe invention include EPO, IFN-α, IFN-β, IFN-γ, consensus IFN, FactorVII, Factor VIII, Factor IX, IL-2, remicade (infliximab), Rituxan(rituximab), Enbrel (etanercept), Synagis (palivizumab), Reopro(abciximab), Herceptin (trastuzimab), tPA, Cerizyme (imiglucerase),Hepatitus-B vaccine, rDNAse, alpha-1 proteinase inhibitor, GCSF, GMCSF,hGH, insulin, FSH, and PTH.

The above exemplary biologically active agents are meant to encompass,where applicable, analogues, agonists, antagonists, inhibitors, isomers,and pharmaceutically acceptable salt forms thereof. In reference topeptides and proteins, the invention is intended to encompass synthetic,recombinant, native, glycosylated, and non-glycosylated forms, as wellas biologically active fragments thereof. The above biologically activeproteins are additionally meant to encompass variants having one or moreamino acids substituted (e.g., cysteine), deleted, or the like, as longas the resulting variant protein possesses at least a certain degree ofactivity of the parent (native) protein.

Pharmaceutical Compositions

The present invention also includes pharmaceutical preparationscomprising a conjugate as provided herein in combination with apharmaceutical excipient. Generally, the conjugate itself will be in asolid form (e.g., a precipitate), which can be combined with a suitablepharmaceutical excipient that can be in either solid or liquid form.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Acids or bases may be present as an excipient in the preparation.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

The pharmaceutical preparations encompass all types of formulations andin particular those that are suited for injection, e.g., powders thatcan be reconstituted as well as suspensions and solutions. The amount ofthe conjugate (i.e., the conjugate formed between the active agent andthe polymer described herein) in the composition will vary depending ona number of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container (e.g., avial). In addition, the pharmaceutical preparation can be housed in asyringe. A therapeutically effective dose can be determinedexperimentally by repeated administration of increasing amounts of theconjugate in order to determine which amount produces a clinicallydesired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, the excipient will be present in the composition inan amount of about 1% to about 99% by weight, preferably from about5%-98% by weight, more preferably from about 15-95% by weight of theexcipient, with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsare described in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andKibbe, A. H., Handbook of Pharmaceutical Excipients, 3^(rd) Edition,American Pharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical preparations of the present invention are typically,although not necessarily, administered via injection and are thereforegenerally liquid solutions or suspensions immediately prior toadministration. The pharmaceutical preparation can also take other formssuch as syrups, creams, ointments, tablets, powders, and the like. Othermodes of administration are also included, such as pulmonary, rectal,transdermal, transmucosal, oral, intrathecal, subcutaneous,intra-arterial, and so forth.

As previously described, the conjugates can be administered injectedparenterally by intravenous injection, or less preferably byintramuscular or by subcutaneous injection. Suitable formulation typesfor parenteral administration include ready-for-injection solutions, drypowders for combination with a solvent prior to use, suspensions readyfor injection, dry insoluble compositions for combination with a vehicleprior to use, and emulsions and liquid concentrates for dilution priorto administration, among others.

Methods of Administering

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with conjugate. The method comprisesadministering, generally via injection, a therapeutically effectiveamount of the conjugate (preferably provided as part of a pharmaceuticalpreparation). The method of administering may be used to treat anycondition that can be remedied or prevented by administration of theparticular conjugate. Those of ordinary skill in the art appreciatewhich conditions a specific conjugate can effectively treat. The actualdose to be administered will vary depend upon the age, weight, andgeneral condition of the subject as well as the severity of thecondition being treated, the judgment of the health care professional,and conjugate being administered. Therapeutically effective amounts areknown to those skilled in the art and/or are described in the pertinentreference texts and literature. Generally, a therapeutically effectiveamount will range from about 0.001 mg to 100 mg, preferably in dosesfrom 0.01 mg/day to 75 mg/day, and more preferably in doses from 0.10mg/day to 50 mg/day.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

One advantage of administering the conjugates of the present inventionis that individual water-soluble polymer portions can be cleaved off.Such a result is advantageous when clearance from the body ispotentially a problem because of the polymer size. Optimally, cleavageof each water-soluble polymer portion is facilitated through the use ofphysiologically cleavable and/or enzymatically degradable linkages suchas urethane, amide, carbonate or ester-containing linkages. In this way,clearance of the conjugate (via cleavage of individual water-solublepolymer portions) can be modulated by selecting the polymer molecularsize and the type functional group that would provide the desiredclearance properties. One of ordinary skill in the art can determine theproper molecular size of the polymer as well as the cleavable functionalgroup. For example, one of ordinary skill in the art, using routineexperimentation, can determine a proper molecular size and cleavablefunctional group by first preparing a variety of polymer derivativeswith different polymer weights and cleavable functional groups, and thenobtaining the clearance profile (e.g., through periodic blood or urinesampling) by administering the polymer derivative to a patient andtaking periodic blood and/or urine sampling. Once a series of clearanceprofiles have been obtained for each tested conjugate, a suitableconjugate can be identified.

All articles, books, patents, patent publications and other publicationsreferenced herein are incorporated by reference in their entireties.

EXAMPLES

It is to be understood that while the invention has been described inconjunction with certain preferred specific embodiments thereof, theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

Abbreviation.

-   DCM: dichloromethane-   NMR: nuclear magnetic resonance-   DI: deionized-   r.t. room temperature-   anh. Anhydrous-   Da Daltons-   GPC gel permeation chromatography    Materials and Methods.

All chemical reagents referred to in the appended examples arecommercially available unless otherwise indicated.

All PEG reagents referred to in the appended examples are available fromNektar, Huntsville, Ala. All ¹HNMR data was generated by a 300 or 400MHz NMR spectrometer manufactured by Bruker.

Example 1 Branched PEG2-amidopentamethylene-maleimide (40 kDa) (L1-AMPE)

Overview of Synthesis:

A. Branched PEG2(40K)Amine

To a solution of branched PEG2(40,000)-N-hydroxysuccinimide ester (20 g,0.00050 moles) (Nektar, Huntsville Ala.) in methylene chloride (250 ml),ethylenediamine (0.68 ml, 0.01017 moles) was added and the reactionmixture was stirred overnight at room temperature under argonatmosphere. Next the solvent was evaporated to dryness. The crudeproduct was dissolved in small amount of methylene chloride andprecipitated with isopropyl alcohol. The wet product was dried underreduced pressure. Yield 17.2 g.

NMR (d₆-DMSO): 2.65 ppm (t, —CH ₂—NH₂), 3.24 ppm (s, —OCH₃), 3.51 ppm(s, PEG backbone).

B. Branched PEG2-amidopentamethylene-maleimide-40 kDa

To a solution of N-succinimidyl(ε-maleimidohexanoate) (0.1 g, 0.000324moles, Pierce Chemical Company), in methylene chloride (10 ml), wasadded over a period of 3 minutes a solution of branched PEG2 (40K) aminefrom Step A (12.2 g, 0.000305 moles) in methylene chloride (20 ml).Next, 0.045 ml of triethylamine was added and the mixture was stirredovernight at room temperature under an argon atmosphere. The solvent wasthen distilled and the crude product that remained was dissolved in 30ml of methylene chloride and then precipitated by the addition of 450 mlof isopropyl alcohol at room temperature. The yield was 11.5 g.

Proton NMR analysis indicated main signals at: 3.24 ppm (s, —OCH₃), 3.51ppm (s, PEG backbone), 7.01 ppm (s, CH═CH, maleimide), which areindicative of the correct product. On the basis of the NMR, thesubstitution was estimated to be approximately 89%. GPC analysisrevealed the main product to be 98.3% desired compound, with 1.7% dimer.The product also possessed required ultraviolet absorption.

Example 2 mPEG(5,000 Da)-butylmalemide (L3-TEME)

Overview of Synthesis:

A. mPEG(5,000 Da)-butylamine

A solution of mPEG-5,000 Da (2.0 g, 0.0004 moles) (NOF Corporation) intoluene (30 ml) was azeotropically dried by distilling off 15 mltoluene. 1.0 M solution of potassium tert-butoxide in tert-butanol (2.0ml, 0.002 moles) and 1,4-dibromobutane (0.43 g, 0.002 moles) were addedand the mixture was stirred overnight at 75° C. under argon atmosphere.The mixture was filtered and the solvents were distilled off underreduced pressure. The residue was dissolved in dichloromethane (3 ml)and isopropyl alcohol (50 ml) was added. The precipitated product wasfiltered off and dried under reduced pressure. Next it was dissolved inconcentrated ammonia (20 ml) and the resulting solution was stirred 20 hat room temperature. The product was extracted with dichloromethane. Theextract was dried with anhydrous magnesium sulfate and the solvent wasdistilled off under reduced pressure giving 1.5 g ofM-PEG(5,000)-butylamine.

NMR (D₂O): 1.53 ppm (m, —CH₂ —CH ₂—CH₂—NH₂) 2.75 ppm (t, —CH ₂—NH₂),3.27 ppm (s, —OCH₃), 3.53 ppm (s, PEG backbone).

B. mPEG(5000 Da)-butylmaleimide

mPEG(5,000)-butylamine (1.0 g, 0.0002 moles) from Step A was dissolvedin saturated aqueous NaHCO₃ (5 ml) and the mixture was cooled to ° C.N-methoxycarbonylmaleimide (0.25 g) was added with vigorous stirring.After stirring for 15 minutes, water (8 ml) was added and the mixturewas stirred an additional 65 minutes. NaCl (0.5 g) was added and the pHwas adjusted to 3.0 with 10% phosphoric acid. The product was extractedwith dichloromethane. The extract was dried with anhydrous MgSO₄ and thesolvent was distilled off under reduced pressure giving 0.9 g of white,solid product.

NMR (d₆-DMSO): 1.48 ppm (bm, —CH₂ —CH ₂—CH₂-Mal), 3.24 ppm (s, —OCH₃),3.51 ppm (s, PEG backbone), 7.00 ppm (s, —CH═CH—).

The product had 82% substitution of the maleimidyl group on the PEGmoiety.

Example 3 mPEG(5000 Da)-hexylmaleimide (L3-HEXA)

This synthesis is essentially equivalent to that described in Example 2above, with the exception that the dibromo-reagent utilized possessestwo additional methylenes, i.e., Br—(CH₂)₆—Br.

A. mPEG(20,000 Da)-hexylamine

A solution of mPEG-5,000 Da (2.0 g, 0.0004 moles) (NOF Corporation) intoluene (30 ml) was azeotropically dried by distilling off 15 mltoluene. 1.0M solution of potassium tert-butoxide in tert-butanol (2.0ml, 0.002 moles) and 1,6-dibromohexane (0.49 g, 0.002 moles) were addedand the mixture was stirred overnight at 80° C. under argon atmosphere.The mixture was filtered and the solvents were distilled off underreduced pressure. The residue was dissolved in dichloromethane (3 ml)and isopropyl alcohol (50 ml) was added. The precipitated product wasfiltered off and dried under reduced pressure. Next it was dissolved inconcentrated ammonia (20 ml) and the resulting solution was stirred 20 hat room temperature. The product was extracted with dichloromethane. Theextract was dried with anhydrous magnesium sulfate and the solvent wasdistilled off under reduced pressure giving 1.6 g ofM-PEG(5,000)-hexylamine.

NMR (D₂O): 1.28 ppm (m, —CH₂ —CH ₂—CH₂—CH₂—NH₂), 1.47 ppm (m, —CH ₂—CH ²—CH₂—CH ₂—CH₂—NH₂), 2.71 ppm (t, —CH ₂—NH₂), 3.27 ppm (s, —OCH₃), 3.53ppm (s, PEG backbone).

B. mPEG(5000 Da)-hexylmaleimide

mPEG(5,000 Da)-hexylamine (1.0 g, 0.0002 moles) from Step A wasdissolved in saturated aqueous NaHCO₃ (5 ml) and the mixture was cooledto ° C. N-methoxycarbonylmaleimide (0.25 g) was added with vigorousstirring. After stirring for 15 minutes, water (8 ml) was added and themixture was stirred an additional 65 minutes. NaCl (0.5 g) was added andthe pH was adjusted to 3.0 with 10% phosphoric acid. The product wasextracted with dichloromethane. The extract was dried with anhydrousMgSO₄ and the solvent was distilled off under reduced pressure giving0.9 g of white, solid product.

NMR (d₆-DMSO): 1.24 ppm (bm, —CH₂ —CH ₂—CH₂—CH₂-Mal), 1.45 ppm (bm, —CH₂—CH₂—CH₂—CH ₂—CH₂-Mal), 3.24 ppm (s, —OCH₃), 3.51 ppm (s, PEG backbone),7.01 ppm (s, —CH═CH—).

The product had 80% substitution of the maleimidyl group on the PEGmoiety.

Example 4 mPEG (5K Da)-propylmaleimide (L3-TME)

Overview of Synthesis:

A. mPEG (5K Da)-Propylamine

To a solution of 4,7,10-trioxa-1,13-tridecanediamine (4.2 g) in anh.acetonitrile (100 ml) was added mPEG-benzotriazolylcarbonate (5 g)(Shearwater Corp.) in anh. acetonitrile (60 ml) during 20 min and themixture was stirred overnight at room temperature under argonatmosphere. Next the solvent was distilled off. The product wasdissolved in 100 ml DI H₂O. NaCl (5 g) was added and the pH was adjustedto 3.0 with 10% H₃PO₄. The product was extracted with CH₂Cl₂. Theextract was washed with 50 ml 2% KOH solution, then it was dried (MgSO₄)and the solvent was distilled off. Next the product was dissolved in 10ml CH₂Cl₂ and reverse precipitated with 200 ml isopropyl alcohol at 0-5°C. Yield after drying 4.2 g.

NMR: Desired product, substitution 85.0%, GPC (buffer, 25° C.)substitution 97.02%.

B. mPEG (5K Da)-PA-Maleimide

mPEG (5K Da)-propylamine (4.0 g in 20 ml deionized water, pH of 8.93)from Step A was cooled to 0-5° C. on an ice bath and a solution ofN-methoxycarbonylmaleimide (0.5 g in 3.5 ml of anh. acetonitrile) wasadded and the mixture was stirred 15 min at 0-5° C. The ice bath wasremoved and DI H₂O (16 ml) was added and the mixture was stirred 45 minat room temperature. NaCl (2 g) was added and the pH of the mixture wasadjusted to 3.0 with 10% H₃PO₄. The product was extracted with CH₂Cl₂.The extract was dried with MgSO₄ and the solvent was distilled off. Thecrude product was dissolved in CH₂Cl₂ (10 ml) and precipitated withisopropyl alcohol (200 ml) at 0-5° C. Yield 3.7 g.

NMR: Confirmed synthesis of desired product; substitution 83.5%.

Example 5 mPEG (5K Da)-amidocyclohexylmethyl-maleimide (L1-MCH)

Overview of Synthesis:

To a solution of 4-(maleimidomethyl)-1-cyclohexanecarboxylic acid, NHSester (0.100 g) (Pierce Chemical Company) in CH₂Cl₂ (10 ml) was added asolution of mPEG (5K Da)-amine (1.5 g) (Shearwater Corp.) in CH₂Cl₂ (20ml). TEA (0.042 ml) was added and the mixture was stirred overnight atroom temperature under argon atmosphere. The solvent was distilled off.The crude product was dissolved in 2 ml CH₂Cl₂ and precipitated withisopropyl alcohol (60 ml) at 0-5° C. Yield 1.35 g.

NMR: Desired compound, substitution 79.7%. GPC (nitrate buffer, 25° C.):dimer: 3.98%; Main compound: 96.02%.

Example 6 Conjugate of mPEG (5K Da)-propylmaleimide (L3-TME)

To illustrate reaction of a reactive polymer of the invention with amolecule bearing a thiol group, to a solution of MPEG-PA-MAL fromExample 4 (1.0 g) in phosphate buffer was added 25 μl of2-mercaptoethanol. The mixture was stirred overnight at room temperatureunder argon atmosphere. The product was extracted with CH₂Cl₂ (3×20 ml).The extract was dried (MgSO4) and the solvent was distilled off. Thecrude product was dissolved in 2 ml CH₂Cl₂ and precipitated with 40 mlisopropyl alcohol at 0-5° C. Yield 0.78 g

NMR: Formation of the desired product was confirmed by NMR;substitution: 64.9%

Example 7 Hydrolysis Rate Study of Reactive Polymers

Using HPLC analysis, the rate of hydrolytic degradation of the maleimidering of several exemplary maleimide-terminated MPEG polymers (averagemolecular weight 5000 Da) was explored.

The following linkages between the maleimide and the PEG polymer segmentwere evaluated: amidoethylene (L1-AMDE), amidopentamethylene (L1-AMPE),amidocyclohexylmethyl (L1-MCH), oxybutyl (L3-TEME), oxyhexyl (L3-HEXA),oxyethyl (L3-ET), and oxypropyl (L3-TME). Complete structures areprovided below for ease of reference.

TABLE 2 Hydrolysis Rates of mPEG (5 k-Da) Maleimides (5 mg/ml) in 50 mMPhosphate Buffer (pH~7.5) as Measured by UV Absorption at 297 nm

mPEG-AMDE-MAL

mPEG-AMPE-MAL

mPEG-MCH-MAL

mPEG-TEME-MAL

mPEG-HEXA-MAL

mPEG-ET-MAL

mPEG-TME-MAL Polymer Structure (Table 1) Half-life (hrs) RelativeHydrolysis Rate mPEG-AMDE-MAL L1-AMDE 8.8 3.66 mPEG-AMPE-MAL L1-AMPE19.4 1.66 mPEG-MCH-MAL L1-MCH 16.3 1.98 mPEG-TEME-MAL L3-TEME 19.6 1.65mPEG-HEXA-MAL L3-HEXA 32.3 1.00 mPEG-ET-MAL L3-ET 8.1 4.01 mPEG-TME-MALL3-TME 11.5 2.82

As can be shown by the data in Table 2 above, the hydrolysis rates ofthese illustrative polymeric maleimides to form their respectivemaleamic acids varies with changes in the structure of the hydrocarbonportion adjacent to the maleimide ring. The data in column threedemonstrates rates of hydrolysis relative to the hexamethylene-maleimidepolymer. As can be seen, for the polymers examined, the L3-HEXA polymerwas the most stable, that is to say, had the slowest hydrolysis rate,and thus, the longest half life. The data above indicates that anincrease in the length of the hydrocarbon chain separating the polymerand the maleimide increases the half-life of the maleimide-terminatedpolymer itself.

Example 8 Hydrolysis Rate Study of Polymer Conjugates

The hydrolysis rates of representative protein and small molecule modelconjugates were investigated to examine the correlation between the ringopening tendencies of the polymer-terminated maleimides themselvesversus their conjugates.

Since large biomolecular components such as proteins have a dramaticeffect on the retention of conjugated molecules on common liquidchromatography columns, it is generally more difficult to measurekinetics of maleimide conjugates than it is for the polymers themselves.In this analysis, the open acid form of the maleamic acid was notdistinctly separable from the unopened or closed ring form. However, acombination analysis based upon size exclusion chromatography (HPLC-SE)and analytical protein electrophoresis (SDS-PAGE) was successfullyemployed to estimate the ring opening characteristics of polymericmaleimide protein conjugates, as well as conjugates prepared using modelnon-protein compounds.

In this study, two PEG-globular protein conjugates represented generallybelow were studied to examine their ring opening characteristics.

The top structure is a PEG-maleimide conjugate of Glob Protein 2, whereGlob Protein 2 is a protein having a molecular weight of approximately48 kDa. Glob Protein 2 was conjugated to a PEG maleimide derived from aPEG propionic acid, MW 30 kDa, which further included a medium-lengthlinker interposed between the propionic acid derived portion of thepolymer and the maleimide terminus. The linker in the top structure is—C(O)—NH(CH₂)₂—NH—C(O)—CH₂CH₂—.

The bottom structure is a PEG-maleimide conjugate of Glob Protein 1,where the protein possesses a molecular weight of about 11 kDa. Theconjugate was prepared using a linkerless maleimide (mPEG-ET-MAL) havinga molecular weight of about 20 kDa. The corresponding PEG maleimidestructure is L3-ET, discussed in Example 7.

The bottom structure (Glob Protein 2) is completely ring opened after 24hours at pH 8.5 at room temperature, thus indicating the instability ofthis type of maleimidyl terminated polymer absent a stabilizing linkerseparating the polymer and the maleimide ring. Relative to thelinkerless form, however, the linker in the top structure (GlobProtein 1) retards the ring opening, since the ring structure in the topconjugate is not completely ring-opened until 17 hours, at pH 9, uponheating to 50° C. for 17 hours.

Similarly, hydrolysis rates of the stabilized polymer maleimides of theinvention conjugated to a model compound, 2-mercaptoethanol, weredetermined to assess the tendency of the conjugates towardsring-opening. The study revealed that the present stabilized polymermaleimides are superior to those used to form conjugates of Glob Protein1 and Glob Protein 2. That it to say, in both free and in conjugatedform, the polymer maleimides of the invention exhibited superiorstability and resistance against ring opening in comparison tolinkerless PEG maleimide and, for example, the PEG-maleimide above,shown attached to Glob Protein 2.

Hydrolysis rate studies of conjugates of 8-TRI, 8-PEN, and 8-MCH(structures provided below in Table 3) were conducted as described abovefor the unconjugated maleimides. The half-lives shown were calculatedfrom data taken at two different pH values. Similar to the unconjugatedmaleimides, the data indicate a slowing in reaction rate as the pHsdrifted lower with increased ring opening. The linkage with the shortesthydrocarbon chain adjacent to the succinimide ring (i.e., 8-TRI) was thefastest to open in comparison to the other conjugates studied. The dataindicate that longer/larger hydrocarbon chains provide superiorresistance to hydrolysis-induced ring opening.

TABLE 3 Hydrolysis Half-lives of mPEG (5 k-Da) Maleimide Conjugates

Experimentally Determined Half-lives Linker, D pH 9.06 pH 8.11 8-TRI; D= trimethylene 31.4 hours 17.6 days 8-PEN; D = pentamethylene — 28.5days

43.3 hours —

Example 9 Synthesis of 1-(N-maleimidomethyl)-4-(methoxypoly(EthyleneGlycol)Propionamidomethyl)Cyclohexane(Mixture of CIS and Trans isomers)

Step 1.

9A. Preparation of 1-aminomethyl-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and transisomers): N-hydroxysuccimidyl ester of methoxypoly(ethyleneglycol)propionic acid, MW 5,000 (20.0 g, 4.0 mmol, Nektar Therapeutics)in acetonitrile (200 mL) was added dropwise to a solution of1,4-cyclohexane(bismethylamine) (11.34 g, 79.7 mmol) in acetonitrile(200 mL) containing triethylamine (20 mL). This mixture was stirred atroom temperature for 3 days. The solvent was removed in vacuo leaving awhite solid. The solid was stirred with ether (100 mL), collected byfiltration and dried to yield 20.23 g of a crude product. This crudemixture was taken up in CH₂Cl₂ (30 mL) and precipitated with IPA (500mL)/ether (250 mL). The solid was collected by filtration and driedunder vacuum (16.3 g).

¹H NMR (dmso-d₆) δ 7.76 (1H, d, NHC═O), 3.51 (br s, O—CH₂CH₂—, PEGbackbone), 2.98 and 2.88 (2H, t, CH ₂—NH—C═O), 2.43 and 2.36 (2H, d, CH₂—NH₂), 2.30 (2H, CH₂C═O), 1.78-1.68 (1H, m, ring CH), 1.45-1.21 (6H, m,ring methylene protons), 0.90-0.73 (1H, m, ring CH).

Step 2.

9.B. Preparation of 1-(N-maleimidomethyl)-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and transisomers): A solution of 1-aminomethyl-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and trans isomers)(3.68 g, 0.74 mmol) in NaHCO₃ (sat'd, 19 mL) was cooled in anice/salt/water bath. To this was added N-methoxycarbonylmaleimide (116mg, 0.82 mmol). This mixture was stirred in the ice bath for 15 minutesand H₂O (29 mL) was added. After stirring in the ice bath for 1 h, thereaction mixture was removed from the bath and stirred at RT for 3 h.The reaction mixture was diluted with brine (30 mL). The pH was adjustedto 3 with 10% phosphoric acid and extracted with CH₂Cl₂ (3×50 mL). Thecombined organic extracts were dried (Na₂SO₄) and concentrated in vacuo.The residue was taken up in CH₂Cl₂ (10 mL) and precipitated with IPA (60mL)/ether (100 mL). The product was collected by filtration and driedunder vacuum overnight (3.14 g).

¹H NMR (dmso-d₆) δ 7.79 (1H, d, NHC═O), 7.01 (2H, s, CH═CH), 3.51 (br s,O—CH₂CH₂—, PEG backbone), 2.32 (2H, CH₂C═O), 1.78-1.45 (2H, m, ringmethylene), 1.45-1.21 (5H, m, ring methylene protons and CH), 0.90-0.73(1H, m, ring protons).

Example 10 Synthesis of Trans-4-(Methoxypoly(EthyleneGlyocol)Propionamidomethyl)-N-cyclohexylmaleimide.

Step 1.

10.A. Preparation of trans-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexyl-t-BOC amine: To a solution oftrans-4-aminomethylcyclohexyl-t-BOC-amine (1.0 g, 4.67 mmol, AlbanyMolecular) and N-hydroxysuccimidyl ester of methoxypoly(ethyleneglycol)propionic acid, MW 5,000 (22.9 g, 4.20 mmol, Nektar Therapeutics)in Acetonitrile (200 mL) was added triethylamine (1.2 mL, 8.6 mmol)under Argon. This mixture was stirred at room temperature under an argonatmosphere for 24 h. NMR did not show any remaining protons from the SPAgroup. The solvent was removed in vacuo to give a white residue whichwas taken up in CH₂Cl₂ (60 mL) and precipitated with IPA (500 mL)/ether(1 L). The solid was collected by filtration and dried under vacuum toyield the product as a white solid (22.2 g).

¹H NMR (dmso-d₆) δ 7.80 (1H, s, CH—NH), 6.70 (1H, d, CH—NH), 3.55 (br s,O—CH₂CH₂—, PEG backbone), 3.28 (3H, s, CH₃), 3.15 (1H, br s, CH), 2.90(2H, t, CH—CH ₂—NH), 2.33 (3 H, t, CH₂—C═O), 1.74-1.65 (4H, M, ringprotons), 1.37 (9 H, s, C(CH₃)₃), 1.25 (1H, br s, CH), 1.14-0.83 (4 H,m, ring protons).

Step 2.

10.B. Preparation of trans-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexylamine, trifluoroacetate. To asolution of trans-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexyl-t-BOC amine (1.20 g, 0.24 mmol) inanhydrous CH₂Cl₂ (5.0 mL) was added trifluoroacetic acid (2.5 mL, 32.5mmol). This mixture was stirred at 25° C. for 18 h. The solvent wasremoved in vacuo leaving an oily residue which was dried overnight undervacuum. The residue was stirred with anhydrous ether (20 mL). Theproduct was collected by filtration followed by drying (0.99 g).

¹H NMR (dmso-d₆) δ 8.13 (1H, br s, NH), 7.80 (3 H, d, NH₃), 3.51 (br s,O—CH₂CH₂—, PEG backbone and CH), 3.24 (3 H, s, CH₃), 2.97 (1 H, br s,CH), 2.27 (2H, t, CH₂C═O), 1.95-1.35 (2H, m, cyclohexane protons),1.34-1.25 (2H, m, cyclohexane protons), 1.40-1.25 (2H, m, cyclohexaneprotons), 1.25-1.13 (2H, m, cyclohexane protons).

Step 3.

10.B. Preparation of trans-4-(Methoxypoly(ethyleneglycol)propionamidomethyl)-N-cyclohexylmaleimide.trans-4-(Methoxypoly(ethylene glycol)propionamidomethyl)cyclohexylamine,trifluoroacetate (3.0 g, 0.60 mmol) was taken up in NaHCO₃ (aq, sat'd,16 mL) and cooled to 2° C. in an ice/salt bath. To this was addedN-methoxycarbonyl maleimide (100 mg, 0.70 mmol). After stirring at 2° C.for 15 minutes, H₂O (24 mL) was added to the reaction mixture andstirring was continued for 4 h. Brine (50 mL) was added followed by pHadjustment to 3 using 10% Phosphoric acid. This mixture was extractedwith CH₂Cl₂ (3×50 mL). The combined organic extracts were dried(Na₂SO₄), concentrated in vacuo, and dried under vacuum. ¹H NMR showedthe product to be ca. 50% maleimide/50% ring-opened material, fromincomplete ring closure.

¹H NMR (dmso-d₆) δ 7.80 (1H, d, NH), 6.96 (2 H, s, maleimide CH═CH),3.51 (474, br s, PEG backbone and CH), 3.24 (3H, s, CH₃), 2.89 (2H, t,CH₂), 2.30 (2H, t, CH₂C═O), 1.85-1.73 (2H, m, cyclohexane protons),1.73-1.63 (2H, m, cyclohexane protons), 1.32 (1H, br s, CH), 1.15-1.05(2H, m, cyclohexane protons), 1.00-0.85 (2H, m, cyclohexane protons).

Example 11 Synthesis of Trans-4-(Methoxypoly(EthyeneGlyol)Propionamido)-N-Cyclohexylmaleimide.

Step 1.

11.A. Preparation of trans-4-(methoxypoly(ethyleneglycol)propionamido)cyclohexyl-t-BOC amine: To a solution ofmono-t-BOC-trans-1,4-diaminocyclohexane (1.0 g, 4.38 mmol, AlbanyMolecular) and N-hydroxysuccimidyl ester of methoxypoly(ethyleneglycol)propionic acid, MW 5,000 (21.5 g, 4.30 mmol, Nektar Therapeutics)in acetonitrile (200 mL) was added triethylamine (1.2 mL, 8.6 mmol)under Argon. This mixture was stirred at room temperature under an argonatmosphere for 24 h. ¹H NMR did not show any remaining protons from theN-hydroxysuccinimidyl ester group. The solvent was removed in vacuo togive a white residue which was stirred with ether (50 mL) for 30minutes. The solid was collected by filtration and dried under vacuum toyield the product as a white solid (1.95 g).

¹H NMR (dmso-d₆) δ 7.70 (1H, d, CH—NH), 6.69 (1H, d, CH—NH), 3.51 (br s,O—CH₂CH₂—, PEG backbone and CH), 3.24 (3 H, s, CH₃), 3.15 (1H, br s,CH), 2.26 (3 H, t, CH₂—C═O), 1.74 (4 H, br d, ring protons), 1.37 (9 H,s, C(CH₃)₃), 1.17-1.07 (4 H, m, ring protons).

Step 2.

11.B. Preparation of trans-4-(methoxypoly(ethyleneglycol)propionamido)cyclohexylamine, trifluoroacetate. To a solution oftrans-4-(methoxypoly(ethylene glycol)propionamido)cyclohexyl-t-BOC amine(12.0 g, 2.4 mmol) in anhydrous CH₂Cl₂ (55 mL) was added trifluoroaceticacid (25 mL, 325 mmol). This mixture was stirred at 25° C. for 18 h. Thesolvent was removed in vacuo leaving an oily residue which was driedovernight under vacuum. The residue was taken up in CH₂Cl₂ (30 mL) andprecipitated with IPA (750 mL)/ether (500 mL). The product was collectedby filtration followed by drying to give the product as a white solid(10.2 g).

¹H NMR (dmso-d₆) δ 8.13 (1H, br s, NH), 7.80 (3 H, d, NH₃), 3.51 (br s,O—CH₂CH₂—, PEG backbone and CH), 3.24 (3 H, s, CH₃), 2.97 (1 H, br s,CH), 2.27 (2H, t, CH₂C═O), 1.95-1.35 (2H, m, cyclohexane protons),1.34-1.25 (2H, m, cyclohexane protons), 1.40-1.25 (2H, m, cyclohexaneprotons), 1.25-1.13 (2H, m, cyclohexane protons).

Step 3.

11.C. Preparation of trans-4-(Methoxypoly(ethyleneglycol)propionamido)-N-cyclohexylmaleimide.trans-4-(Methoxypoly(ethylene glycol)propionamido)cyclohexylamine,trifluoroacetate (3.0 g, 0.60 mmol) was taken up in NaHCO₃ (aq, sat'd,16 mL) and cooled to 2° C. in an ice/salt bath. To this was addedN-methoxycarbonyl maleimide (100 mg, 0.70 mmol). After stirring at 2° C.for 15 minutes, H₂O (24 mL) was added to the reaction mixture andstirring was continued for 5 h. Brine (20 mL) was added followed by pHadjustment to 3 using 10% Phosphoric acid. This mixture was extractedwith CH₂Cl₂ (3×50 mL). The combined organic extracts were dried(Na₂SO₄), concentrated in vacuo, and dried under vacuum to give theproduct as a white solid (2.75 g). ¹H NMR showed the product to be 26%maleimide/74% opened material, from incomplete ring closure.

¹H NMR (dmso-d₆) δ 7.77 (1H, d, NH), 6.96 (2 H, s, maleimide CH═CH),3.51 (br s, O—CH₂CH₂—, PEG backbone), 3.24 (3H, s, CH₃), 2.28 (2H, t,CH₂C═O), 2.06-1.92 (1H, m, cyclohexane proton), 1.88-1.73 (3H, m,cyclohexane protons), 1.59-1.65 (1H, m, cyclohexane proton), 1.28-1.13(3H, m, cyclohexane protons).

Example 12 Synthesis of 1-N-maleimidomethyl-3-(Methoxypoly(EthyleneGlyol)Propionamidomethyl)Cyclohexane(Mixture of CIS and Trans isomers)

Step 1.

12.A. Preparation of 1-aminomethyl-3-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and transisomers): N-hydroxysuccimidyl ester of methoxypoly(ethyleneglycol)propionic acid, MW 5,000 (10.0 g, 2.0 mmol, Nektar Therapeutics)in acetonitrile (100 mL) was added dropwise to a solution of1,3-cyclohexane(bismethylamine) (6.0 mL, 39.9 mmol, Albany Molecular) inacetonitrile (100 mL) containing triethylamine (10 mL). This mixture wasstirred at room temperature for 3 days. The insoluble solids werefiltered and the filtrate was concentrated in vacuo leaving a whitesolid. The crude mixture was taken up in CH₂Cl₂ (50 mL) and precipitatedwith IPA (375 mL)/ether (300 mL). The solid was filtered to give a whitesolid which was dried under vacuum (9.5 g). ¹H NMR showed some remaining1,3-cyclohexane(bismethylamine) which was removed by dissolving thesolid in CH₂Cl₂ and washing through Amberlyst 15 (15 g). The solvent wasremoved to give 1-methylamino-3-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (8.62 g).

¹H NMR (dmso-d₆) δ 7.78 (1H, d, NHC═O), 3.51 (br s, PEG backbone), 3.24(3H, s, OCH₃), 2.95 and 2.89 (2H, m, CH ₂—NH—C═O), 2.49 and 2.37 (2H, d,CH ₂—NH₂), 2.30 (2H, CH₂C═O), 1.75-1.65 (4H, m, ring protons), 1.63-1.21(2H, m, ring protons), 1.21-1.03 (2H, m, ring protons), 0.80-0.70 (1H,m, ring proton), 0.50-0.30 (1H, m, ring proton).

Step 2.

12.B. Preparation of 1-N-maleimidomethyl-3-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and transisomers): A solution of 1-aminomethyl-3-(methoxypoly(ethyleneglycol)propionamidomethyl) cyclohexane (mixture of cis and transisomers) (4.0 g, 0.8 mmol) in NaHCO₃ (sat'd, 20 mL) was cooled in anice/salt/water bath. To this was added N-methoxycarbonylmaleimide (126mg, 0.89 mmol). This mixture was stirred in the ice bath for 15 minutesand H₂O (32 mL) was added. After stirring in the ice bath for 1 h, thereaction mixture was removed from the bath and stirred at RT for 3 h.The reaction mixture was diluted with brine (30 mL). The pH was adjustedto 3 with 10% phosphoric acid and extracted with CH₂Cl₂ (3×50 mL). Thecombined organic extracts were dried (Na₂SO₄) and concentrated in vacuo.The residue was dried under vacuum to give the product as a white solid.

¹H NMR (dmso-d₆) δ 7.79 (1H, d, NHC═O), 7.01 (2H, s, CH═CH), 3.51 (br s,PEG backbone and CHCH ₂), 3.24 (3H, s, OCH₃) 3.10-2.63 (2H, m, CHCH ₂),2.31 (2H, dd CH₂C═O), 1.65-1.1.10 (8H, m, ring protons), 0.83-0.48 (2H,m, ring protons).

Example 13 Hydrolysis Rate Study of Exemplary Polymer Maleimides

Hydrolysis studies were conducted on several exemplary stabilizedPEG-maleimides as described above in Example 7. The structures of theparticular PEG-maleimides and their corresponding half-lives areprovided in Table 4 below.

TABLE 4 Stability of Selected mPEG Maleimides STRUCTURE HALF-LIFE, INHOURS

8.1

11.5

20.6

64.6

Example 14 Conjugation of a Stabilized mPEG-maleimide,1-(N-maleimidomethyl)-4-(Methoxypoly(EthyleneGlycol)Propionamidomethyl)Cyclohexane, to the Model Compound,2-mercaptoethanol

Preparation of1-(3-(2-hydroxyethanemercapto)-N-succinimidylmethyl)-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and trans isomers)To a solution of 1-(N-maleimidomethyl)-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and trans isomers)(500 mg, 0.1 mmol) in CH₃CN (10 mL) was added 2-mercaptoethanol (15 μL,0.21 mmol). This mixture was allowed to stir at room temperature for 18hours. ¹H NMR showed remaining maleimide starting material. Additional2-mercaptoethanol (15 μL, 0.21 mmol) was added and the mixture wasstirred for another 24 hours. ¹H NMR showed that no remaining maleimidewas present. The solvent was removed in vacuo and dried under vacuum.The solid was taken up in CH₂Cl₂ (2 mL) and precipitated with IPA (50mL). The solid was collected by filtration and dried to give the productas a white solid (403 mg).

¹H NMR (dmso-d₆) δ 7.66 (1H, br s, NH), 4.83 (1H, t, OH), 4.01 (1H, dd,CH—S), 3.51 (br s, PEG backbone), 3.05-2.61 (4H, m, 2×CHCH ₂), 2.30 (2H,t, CH₂C═O), 1.75-0.75 (10H, m, ring protons).

Example 15 Conjugation of a Stabilized mPEG-maleimide,Trans-4-(Methoxypoly(EthyleneGlyol)Propionamidomethyl)-N-cyclohexylmaleimide, to the Model Compound,2-mercaptoethanol

Preparation oftrans-1-(3-(2-hydroxyethanemercapto)-N-succinimidyl)-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane: To a solution oftrans-4-(methoxypoly(ethyleneglycol)propionamidomethyl)-N-cyclohexylmaleimide (400 mg, 0.0.08 mmol)in CH₃CN (10 mL) was added 2-mercaptoethanol (15 μL, 0.21 mmol). Thismixture was allowed to stir at room temperature for 18 hours. Thesolvent was removed in vacuo and dried under vacuum. The residue wasstirred with ether (2×20 mL) and the solid collected by filtration togive the product as a white solid (310 mg).

¹H NMR (dmso-d₆) δ 7.81 (1H, br s, NH), 4.87 (1H, t, OH), 3.85 (1H, dd,CH—SEtOH), 3.51 (br s, PEG backbone, CH and SCH₂CH ₂OH), 3.24 (3H, s,CH₃), 2.90 (2H, br s, CH₂NH), 2.79 (2H, t, SCH ₂CH₂), 2.30 (2H, t,CH₂C═O), 2.08-1.55 (5H, m, ring protons), 1.25-0.94 (4H, m, ringprotons).

Example 16 Conjugation of a Stabilized mPEG-maleimide,Trans-4-(Methoxypoly(EthyleneGlycol)Propionamido)-N-cyclohexylmaleimide, to the Model Compound,2-mercaptoethanol

Preparation oftrans-1-(3-(2-hydroxyethanemercapto)-N-succinimidyl)-4-(methoxypoly(ethyleneglycol)propionamido)cyclohexane: To a solution oftrans-4-(methoxypoly(ethylene glycol)propionamido)-N-cyclohexylmaleimide(400 mg, 0.0.08 mmol) in CH₃CN (10 mL) was added 2-mercaptoethanol (15μL, 0.21 mmol). This mixture was allowed to stir at room temperature for18 hours. The solvent was removed in vacuo and dried under vacuum togive the product as a white solid (240 mg).

¹H NMR (dmso-d₆) δ 7.75 (1H, br s, NH), 4.83 (1H, t, OH), 4.01 (1H, dd,CH—S), 3.51 (br s, PEG backbone and 2×CH), 2.75 (2H, m, S—CH₂), 2.33(2H, t, CH₂C═O), 2.02-1.65 (4H, m, ring protons), 1.55-0.95 (4H, m, ringprotons).

Example 17 Conjugation of a Stabilized mPEG-maleimide,1-N-maleimidomethyl-3-(Methoxypoly(EthyleneGlycol)Propionamidomethyl)Cyclohexane, to the Model Compound,2-Mercaptoethanol

Preparation of1-(3-(2-hydroxyethanemercapto)-N-succinimidylmethyl)-3-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and transisomers): To a solution of 1-N-maleimidomethyl-3-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and trans isomers)(450 mg, 0.0.09 mmol) in CH₃CN (10 mL) was added 2-mercaptoethanol (15μL, 0.21 mmol). This mixture was allowed to stir at room temperature for18 hours. The solvent was removed in vacuo and dried under vacuum. Theresidue was stirred with ether (20 mL) and the solid collected byfiltration to give the product as a white solid (230 mg).

¹H NMR (dmso-d₆) δ 7.81 (1H, br s, NH), 4.89 (1H, t, OH), 4.03 (1H, dd,CH—S), 3.51 (PEG backbone and S—CH ₂CH ₂), 3.24 (3H, s, CH₃), 3.05-2.60(4H, m, 2×CHCH ₂), 2.30 (2H, t, CH ₂C═O), 1.80-1.05 (8H, m, ringprotons), 0.80-0.51 (2H, m, ring protons).

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing description.Therefore, it is to be understood that the invention is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims. Although specific terms are employed herein, they areused in a generic and descriptive sense only and not for purposes oflimitation.

1. A conjugate formed by reaction of an active agent comprising areactive thiol group with a water-soluble polymer having the structure:

wherein: POLY is a water-soluble polymer segment, b is 0 or 1, X is ahydrolytically stable linker comprising at least 4 contiguous saturatedcarbon atoms adjacent to the nitrogen atom of the maleimide ring, andsaid polymer is absent aromatic groups and ester linkages.
 2. Aconjugate comprising the following structure:

wherein: POLY is a water-soluble polymer segment, b is 0 or 1, X is ahydrolytically stable linker comprising at least 4 contiguous saturatedcarbon atoms adjacent to the ring nitrogen atom,“POLY-[O]_(b)—C(O)—NH—X—” is absent aromatic groups and ester linkages,and “—S-active agent” represents a residue of an active agent comprisinga thiol (—SH) group.
 3. A composition comprising the conjugate of claim2, wherein said composition comprises a single polymer conjugatespecies.
 4. The conjugate of claim 1, wherein said active agent isselected from the group consisting of small molecules, peptides, andproteins.
 5. The conjugate of claim 2, wherein said active agent isselected from the group consisting of small molecules, peptides, andproteins.
 6. The conjugate of claim 2, wherein POLY is selected from thegroup consisting of poly(alkylene oxide), poly(vinyl pyrrolidone),poly(vinyl alcohol), polyoxazoline, poly(acryloylmorpholine), andpoly(oxyethylated polyol).
 7. The conjugate of claim 6, wherein POLY isa poly(alkylene oxide).
 8. The conjugate of claim 7, wherein POLY is apoly(ethylene glycol).
 9. The conjugate of claim 8, wherein thepoly(ethylene glycol) is terminally capped with an end-capping moiety.10. The conjugate of claim 9, wherein the end-capping moiety is selectedfrom the group consisting alkoxy, substituted alkoxy, alkenyloxy,substituted alkenyloxy, alkynyloxy, substituted alkynyloxy, aryloxy, andsubstituted aryloxy.
 11. The conjugate of claim 10, wherein theend-capping moiety is selected from the group consisting of methoxy,ethoxy, and benzyloxy.
 12. The conjugate of claim 8, wherein thepoly(ethylene glycol) has a nominal average molecular mass of from about100 daltons to about 100,000 daltons.
 13. The conjugate of claim 12,wherein the poly(ethylene glycol) has a nominal average molecular massof from about 1,000 daltons to about 50,000 daltons.
 14. The conjugateof claim 13, wherein the poly(ethylene glycol) has a nominal averagemolecular mass of from about 2,000 daltons to about 30,000 daltons. 15.The conjugate of claim 8, wherein said poly(ethylene glycol) has astructure selected from the group consisting of linear, branched andforked.
 16. The conjugate of claim 15, wherein said poly(ethyleneglycol) comprises the structure:Z—(CH₂CH₂O)_(n)—CH₂CH₂—, where n is from about 10 to about 4000, and Zcomprises a moiety selected from the group consisting of hydroxy, amino,ester, carbonate, aldehyde, aldehyde hydrate, acetal, ketone, ketonehydrate, ketal, alkenyl, acrylate, methacrylate, acrylamide, sulfone,thiol, carboxyl acid, isocyanate, isothiocyanate, hydrazide, urea,maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide,alkoxy, benzyloxy, silane, lipid, phospholipid, biotin, and fluorescein.17. The conjugate of claim 8, wherein X is a saturated acyclic, cyclic,or alicyclic hydrocarbon chain having a total number of carbon atomsselected from the group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, and
 20. 18. The conjugate of claim 17, wherein Xis a saturated acyclic, cyclic, or alicyclic hydrocarbon chain having atotal number of carbon atoms selected from the group consisting of: from4 to about 12, from 4 to about 10, and from about 5 to about 8 atoms.19. The conjugate of claim 8, wherein X is a linear saturated acyclichydrocarbon chain.
 20. The conjugate of claim 8, wherein X is a branchedsaturated acyclic hydrocarbon chain.
 21. The conjugate of claim 20,wherein X is branched at the carbon α to the maleimidyl group.
 22. Theconjugate of claim 20, wherein X is branched at the carbon β to themaleimidyl group.
 23. The conjugate of claim 20, wherein X is branchedat the carbon γ to the maleimidyl group.
 24. The conjugate of claim 19,having the structure:

wherein: y is an integer from 4 to about 20; R¹, in each occurrence, isindependently H or an organic radical that is selected from the groupconsisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl, andR², in each occurrence, is independently H or an organic radical that isselected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl.
 25. The conjugate of claim 24, wherein either (i) R¹and R² in each occurrence is independently H or an organic radicalselected from the group consisting of lower alkyl and lower cycloalkylor (ii) R¹ and R² are both H, and y is selected from the groupconsisting of 4, 5, 6, 7, 8, 9, and
 10. 26. The conjugate of claim 24having the structure:

wherein at least one of R¹ or R² on C_(α) is selected from the groupconsisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl. 27.The conjugate of claim 26, wherein each of R¹ and R² on C_(α) isindependently selected from the group consisting of alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, alkylenecycloalkyl, andsubstituted alkylenecycloalkyl.
 28. The conjugate of claim 26, whereinall other non-C_(α) R¹ and R² variables are H.
 29. The conjugate ofclaim 26 wherein at least one of R¹ or R² onC_(α is lower alkyl or lower cycloalkyl.)
 30. The conjugate of claim 26,wherein R² on C_(α) is H.
 31. The conjugate of claim 30, wherein R¹ onC_(α) is selected from the group consisting of methyl, ethyl, propyl,isopropyl, butyl, isobutyl, pentyl, cyclopentyl, hexyl, and cyclohexyl.32. The conjugate of claim 26 having the structure:

wherein R¹ and R² are each independently alkyl or cycloalkyl.
 33. Theconjugate of claim 26, having the structure:

wherein R¹ is alkyl or cycloalkyl and R² is H.
 34. The conjugate ofclaim 32 wherein R¹ and R² are each independently either methyl orethyl.
 35. The conjugate of claim 32, wherein R¹ and R² are the same.36. The conjugate of claim 22 having the structure:

wherein R¹ and R² are each independently selected from the groupconsisting of H, alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl, butare not both H.
 37. The conjugate of claim 36, wherein R¹ and R² areeach independently H, lower alkyl or lower cycloalkyl.
 38. The conjugateof claim 37, wherein R¹ and R² are each independently selected from thegroup consisting of H, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, pentyl, cyclopentyl, hexyl, and cyclohexyl.
 39. The conjugateof claim 36, wherein R² is H.
 40. The conjugate of claim 24, having thestructure:

wherein at least one of R¹ and R² attached to C_(γ) is selected from thegroup consisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl. 41.The conjugate of claim 40, wherein at least one of R¹ and R² attached toC_(γ is alkyl or cycloalkyl and all other R) ¹ and R² variables are H.42. The conjugate of claim 40, wherein one of the R¹ variables attachedto C_(α) or C_(β) is alkyl or cycloalkyl, and all other R¹ and R²variables are H.
 43. The conjugate of claim 2, wherein X is a saturatedcyclic or alicyclic hydrocarbon chain.
 44. The conjugate of claim 43,wherein X has the structure:

and CYC_(a) is a cycloalkylene group having “a” ring carbons, where thevalue of “a” ranges from 3 to 12; p and q are each independently 0 to20, and p+q+a≦20, R¹, in each occurrence, is independently H or anorganic radical that is selected from the group consisting of alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl,alkylenecycloalkyl, and substituted alkylenecycloalkyl, and R², in eachoccurrence, is independently H or an organic radical that is selectedfrom the group consisting of alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl.
 45. The conjugate of claim 44, wherein p and q areeach independently selected from the group consisting of 0, 1, 2, 3, 4,5, 6, 7, and
 8. 46. The conjugate of claim 44, wherein R¹, in eachoccurrence, is independently H or an organic radical that is eitherlower alkyl or lower cycloalkyl, and R², in each occurrence, isindependently H or an organic radical that is either lower alkyl orlower cycloalkyl.
 47. The conjugate of claim 44, wherein a is selectedfrom the group consisting of 5, 6, 7, 8 and
 9. 48. The conjugate ofclaim 47, wherein a is 6 and CYC_(a) is a 1,1-, 1,2-, 1,3- or1,4-substituted cyclohexyl ring.
 49. The conjugate of claim 44, whereinp and q each independently range from 0 to
 4. 50. The conjugate of claim48, wherein the substituents on said substituted cyclohexyl ring arecis.
 51. The conjugate of claim 48, wherein the substituents on saidsubstituted cyclohexyl ring are trans.
 52. The conjugate of claim 44,wherein R¹ and R² are H in every occurrence.
 53. The conjugate of claim46, having the structure:

wherein q and p each independently range from 0 to
 6. 54. The conjugateof claim 53, wherein q ranges from 0 to 6 and p is zero.
 55. Theconjugate of claim 46, having the structure:

wherein q and p each independently range from 0 to 3, and thesubstituents on the cyclohexylene ring are either cis or trans.
 56. Theconjugate of claim 44, wherein CYC_(a) is bicyclic or tricyclic.
 57. Theconjugate of claim 56, wherein CYC_(a) is selected from the groupconsisting of:

which may include substituents positioned at any available position onthe bi or tricyclic ring.
 58. The conjugate of claim 2 having thestructure:

wherein X and b are as previously defined, b′ is0 or 1, and X′ is ahydrolytically stable linker comprising at least 4 contiguous saturatedcarbon atoms adjacent to the ring nitrogen atom.
 59. The conjugate ofclaim 2, corresponding to the structure:

wherein: d is an integer from 3 to about 100, and R is a residue of acentral core molecule having 3 or more hydroxyl groups, amino groups, orcombinations thereof.
 60. The conjugate of claim 59, wherein d is aninteger from 3 to about
 12. 61. The conjugate of claim 1, wherein POLYis a multi-arm polymer segment, and said polymer corresponds to thestructure:

where PEG is —(CH₂CH₂O)_(n)CH₂CH₂—, M is:

and m is selected from the group consisting of 3, 4, 5, 6, 7, and 8.